Organ regeneration method using ips cells and blastocyst complementation

Abstract

To provide an industrially-suitable technique for organ regeneration using an easily-producible induced pluripotent stem cell (iPS cell).SOLUTION: An organ regeneration method clarifies that in a blastocyst complementation method, organ regeneration can be achieved by utilizing a fact that deficiency in an organ such as pancreas can be complemented by injecting an induced pluripotent stem cell (an iPS cell) into a developed blastocyst, thereby resolving the issue. The method thus uses the iPS cell to produce, in a living body of a nonhuman mammal having abnormality not causing development of a target organ during a development stage, the target organ derived from a different individual mammal that is an individual different from the nonhuman mammal.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a desired cell-derived organ in vivo using iPS cells. [Background technology]

[0002] When discussing regenerative medicine in the form of cell or organ transplantation, there are high expectations for pluripotent stem cells. ES cells, established from the inner cell mass of blastocyst-stage fertilized eggs, have pluripotency and are used in research on various cell differentiation, and the development of differentiation control methods to induce differentiation of ES cells into specific cell lineages in vitro is a hot topic in regenerative medicine research.

[0003] In vitro differentiation studies using ES cells have shown that they readily differentiate into mesodermal and ectodermal lineages, such as blood cells, blood vessels, cardiac muscle, and the nervous system, which differentiate during early embryonic development. However, it is generally known that differentiation into organs, which lead to the formation of complex tissues through cell-cell interactions from the mid-embryonic stage onwards, is difficult.

[0004] For example, the mammalian adult kidney, the metanephros, develops from the intermediate mesoderm during mid-embryonic development. Kidney development begins with the interaction of two components: metanephric mesenchymal cells and ureteric bud epithelium. Ultimately, the adult kidney is completed through the differentiation of dozens of functional cell types, a number not seen in other organs, and the formation of complex nephron structures centered around glomeruli and tubules. Given the timing and complexity of kidney development, it is easy to imagine that inducing kidneys from ES cells in vitro would be an extremely difficult and time-consuming task, and is considered virtually impossible. Furthermore, the identification of somatic stem cells in organs such as the kidney has yet to be conclusively established, and it is becoming clear that the contribution of bone marrow cells, once extensively studied, to the repair process of damaged kidneys is not particularly significant.

[0005] When pluripotent ES cells are injected into the lumen of a blastocyst-stage fertilized egg, the resulting individual forms a chimeric mouse. Previously, a rescue experiment of T cell and B cell lineages using blastocyst complementation using this technology has been reported for Rag-2 knockout mice, which lack T cell and B cell lineages (Non-Patent Document 1). This chimeric mouse assay is used as an in vivo assay system to confirm differentiation of the T cell lineage, for which no in vitro assay system exists.

[0006] However, even if it is proven that such a technology can be used for a certain organ, it is difficult to predict whether it will actually be successful for other organs, as various factors influence the outcome, such as the organ's role in the body, and the lethality of its absence. Furthermore, the deleted genes in the organ defect model selected here are also an important factor, as it is necessary to select transcription factors that function during the developmental process, particularly those essential for the differentiation and maintenance of stem / progenitor cells for each organ during organ formation.

[0007] If this were a model in which organ deficiency was caused by a lack of humoral or secreted factors, it would be expected that only the released factors would be replenished by those released from cells derived from ES cells, resulting in a chimeric state at the organ level.

[0008] For this reason, the selection of an appropriate model animal for the organ is a key factor in this invention, and when considering application to other organs, it is thought to be difficult to use a model that shows a phenotype similar to that of the present invention in other organs.

[0009] The present inventors have filed PCT / JP2008 / 51129 as a method for organ regeneration.

[0010] Recently, induced pluripotent stem (iPS) cells have been attracting attention (for example, Non-Patent Document 2). iPS cells are believed to have functions equivalent to those of ES cells. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Chen J.et al.,Proc.Natl.Acad.Sci.USA,Vol.90,pp.4528-4532,1993 [Non-patent document 2] Okita K et al., Generation of germline-competent induced pluripotent stem cells.Nature 448(7151)313-7, 2007 Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention aims to provide an organ regeneration technology suitable for industrial use using easily prepared induced pluripotent stem cells (iPS cells). Specifically, the present invention aims to provide a technology for regenerating "one's own organs" from somatic cells such as skin, tailored to individual characteristics. Another objective is to carry out research and development using organs derived from various genomes by producing induced pluripotent stem cells (iPS cells) based on cells with a desired genome and practicing the present invention. Another objective is to avoid the ethical issues that have arisen with ES cells. [Means for solving the problem]

[0013] The present invention has discovered that in a blastocyst complementation method, injecting induced pluripotent stem cells (iPS cells) into developed blastocysts to complement missing organs such as the pancreas results in the birth of the next generation, and has also discovered that transgenic animals with a complemented pancreas can act as founders to transmit their phenotype to the next generation. This has demonstrated that organ regeneration can be achieved using such founders, thereby solving the above-mentioned problems.

[0014] In the present invention, it has been discovered that, for example, by transplanting induced pluripotent stem cells (iPS cells) as pluripotent cells into knockout mice and transgenic animals (e.g., mice) characterized by the absence of organs such as the pancreas, it is possible to supplement the pancreas and efficiently produce offspring as founders.

[0015] In the present invention, it was found that even when induced pluripotent stem cells (iPS cells) were used, knockout mice with a complemented pancreas, as a result of genotyping, grew normally to adulthood.

[0016] Compensated knockout (hereinafter referred to as "KO") mice are produced by mating a KO mouse, which acts as a founder and transmits its phenotype to the next generation, with a heterozygous mouse. In theory, according to the laws of Mendelian inheritance, there should be a 50 / 50 chance of producing a KO or heterozygous individual, and we found that this was actually the case. If two KO mice with complemented pancreases are mated, it will be possible to obtain KO individuals in the next generation with 100% success, which is likely to make analysis using KO individuals significantly easier.

[0017] Furthermore, it has been shown that induced pluripotent stem cells (iPS cells) can be used in both the conventional transgenic (Tg) animal production method, in which a transgene that induces organ defects is inserted into egg cells and then transplanted, and in the relatively new method, in which ES cells are injected into the developed blastocysts to compensate for the pancreatic defect and produce the next generation. Furthermore, it has been confirmed that transgenic animals with a complemented pancreas using induced pluripotent stem cells (iPS cells) can also pass on their phenotype to the next generation as founders. Therefore, it has been demonstrated that organ regeneration can also be achieved using induced pluripotent stem cells (iPS cells) using such founders.

[0018] Once it has been shown that the method of the present invention can be applied to a certain organ, it can be understood that the method can be applied to that organ with appropriate modifications based on successful cases. The reason for this is as follows: if an appropriate defective animal exists, by using fluorescently labeled iPS cells (e.g., derived from fibroblasts collected from the skin or tail) as shown herein and applying a similar analytical method, it will become clear whether the constructed organ is derived from the host or iPS cells, etc., and it will be possible to determine whether the organ can be constructed, and it will be understood that, based on the same theory, it will be possible to reproduce the next generation of animals.

[0019] Thus, the present invention provides the following:

[0020] In one aspect, the present invention provides a method for producing a target organ from a heterologous mammal, which is an individual different from a non-human mammal having an abnormality that prevents the development of the target organ at a developmental stage, in the living body of the non-human mammal, the method comprising: a) preparing induced pluripotent stem cells (iPS cells) derived from the heterologous mammal; b) implanting the cells into a blastocyst stage fertilized egg of the non-human mammal; c) allowing the fertilized egg to develop in the womb of a non-human foster mammal to obtain a liveborn offspring; and d) A method for producing a target organ, which comprises the step of obtaining the target organ from the offspring.

[0021] In one embodiment, the iPS cells are derived from a human, a rat, or a mouse.

[0022] In one embodiment, the iPS cells are derived from a rat or a mouse.

[0023] In one embodiment, the organ to be produced is selected from the group consisting of pancreas, kidney, thymus, and hair.

[0024] In one embodiment, the non-human mammal is a mouse.

[0025] In one embodiment, the mouse is a Sall1 knockout mouse, a Pdx1-Hes1 transgenic mouse, a Pdx-1 knockout mouse, or a nude mouse.

[0026] In one embodiment, the target organ is entirely derived from the allogeneic mammal.

[0027] In one embodiment, the method of the present invention further comprises the step of obtaining the iPS cells by contacting somatic cells with reprogramming factors.

[0028] In one embodiment, in the method of the present invention, the iPS cells and the non-human mammal are of different species.

[0029] In one embodiment, in the method of the present invention, the iPS cells are derived from a rat, and the non-human mammal is a mouse.

[0030] In another aspect, the present invention provides a non-human mammal having an abnormality in which development of a target organ does not occur at a developmental stage, comprising: a) preparing iPS cells derived from a heterologous mammal that is an individual different from the non-human mammal; b) implanting the iPS cells into a blastocyst stage fertilized egg of the non-human mammal; and c) providing a mammal produced by a method comprising the step of developing the fertilized egg in the womb of a non-human foster mammal to obtain a litter.

[0031] In another aspect, the present invention relates to the use of a non-human mammal having an abnormality that prevents the development of a target organ at the developmental stage, for producing the target organ using iPS cells.

[0032] In another aspect, the present invention provides a set for producing a target organ, the set comprising: A) a non-human mammal having an abnormality in which the target organ does not develop during the developmental stage; B) A set is provided, comprising iPS cells or reprogramming factors derived from a heterologous mammal that is an individual different from the non-human mammal, and, if necessary, somatic cells.

[0033] In another aspect, the present invention provides a method for producing a desired organ or body part, comprising the steps of: A) providing an animal containing a deficiency-causing gene encoding a cause of deficiency in an organ or body part that would be non-viable or difficult to survive if functional, and the organ or body part is complemented by blastocyst complementation, wherein the deficiency-causing gene encodes a cause of deficiency in the organ or body part of interest; B) obtaining eggs from said animal and developing them into blastocysts; C) introducing into the blastocyst a target iPS cell having a desired genome capable of complementing the defect caused by the defective gene to produce a chimeric blastocyst; and D) producing an individual from the chimeric blastocyst and obtaining the desired organ or body part from the individual.

[0034] In one embodiment, the method of the present invention further comprises the step of obtaining the iPS cells by contacting somatic cells with reprogramming factors.

[0035] In one embodiment, the step D) comprises developing the chimeric blastocyst in the womb of a non-human foster mammal to obtain a baby, and obtaining the target organ from the baby.

[0036] In another embodiment, the target iPS cells are derived from a rat or a mouse.

[0037] In another embodiment, the organ or body part of interest is selected from the group consisting of pancreas, kidney, thymus, and hair.

[0038] In yet another embodiment, the animal is a mouse.

[0039] In another embodiment, the mouse is a Sall1 knockout mouse, a Pdx-1 knockout mouse, a Pdx1-Hes1 transgenic mouse, or a nude mouse.

[0040] In yet another embodiment, the desired organ or body part is entirely derived from the desired pluripotent cells.

[0041] In yet another embodiment, the iPS cells and the non-human mammal are of different species.

[0042] In yet another embodiment, the iPS cells are derived from a rat and the non-human mammal is a mouse.

[0043] In another aspect, the present invention provides a set for producing a desired organ or body part, the set comprising: A) a non-human animal that contains a gene encoding a cause of a defect in an organ or body part that would be non-viable or difficult to survive if it were to function, and that the organ or body part is to be complemented by complementation; and B) a combination of iPS cells or reprogramming factors derived from a xenogeneic mammal that is an individual different from the non-human mammal, and somatic cells as needed.

[0044] In one embodiment, the non-human animal and the iPS cells are of a xenogeneic relationship.

[0045] In the present invention, cells to be transplanted are prepared according to the animal species of the organ to be produced. For example, if a human organ is to be produced, human-derived cells are prepared, and if a non-human mammalian organ is to be produced, cells derived from that mammal are prepared. In the present invention, induced pluripotent stem cells (iPS cells) can be used as the cells to be transplanted.

[0046] The organ to be produced by the method of the present invention may be any solid organ having a specific shape, such as kidney, heart, pancreas, cerebellum, lung, thyroid, hair, and thymus, but preferably includes kidney, pancreas, hair, and thymus. Such solid organs are produced in the body of the offspring by developing totipotent or pluripotent cells in a recipient embryo. Because totipotent or pluripotent cells can form all organs when developed in an embryo, the type of solid organ that can be produced does not depend on the type of totipotent or pluripotent cells used.

[0047] On the other hand, the present invention is characterized by forming an organ derived solely from the transplanted cells in the body of an offspring derived from a recipient non-human embryo, and it is undesirable to have a chimeric cellular structure between the cells derived from the recipient non-human embryo and the transplanted cells. Therefore, it is desirable to use, as the recipient non-human embryo, an embryo derived from an animal with an abnormality in which the organ to be produced does not develop during the developmental stage and the offspring will be deficient in that organ. The animal that will develop such organ defects may be a knockout animal that develops an organ defect due to the deficiency of a specific gene, or a transgenic animal that develops an organ defect due to the incorporation of a specific gene. Alternatively, it may be a "founder" animal as described herein.

[0048] For example, when producing a kidney as an organ, the recipient non-human embryo can be an embryo of a Sall1 knockout animal (Nishinakamura, R. et al., Development, Vol. 128, p. 3105-3115, 2001), which has an abnormality in which kidneys do not develop during the developmental stage. Furthermore, when producing a pancreas as an organ, the recipient non-human embryo can be an embryo from a Pdx-1 knockout animal (Offield, M.F., et al., Development, Vol. 122, pp. 983-995, 1996), which has an abnormality in which the pancreas does not develop during development. When producing a cerebellum as an organ, the recipient non-human embryo can be an embryo from a Wnt-1 (int-1) knockout animal (McMahon, A.P. and Bradley, A., Cell, Vol. 62, pp. 1073-1085, 1990), which has an abnormality in which the cerebellum does not develop during development. When producing a lung or thyroid as an organ, the recipient non-human embryo can be an embryo from a T / ebp knockout animal (Kimura, S., et al., Genes and Development, Vol. 10, pp. 60-69, 1996), which has an abnormality in which the lung and thyroid do not develop during development. Alternatively, embryos from a dominant-negative transgenic mutant animal model (Celli, G., et al., EMBO J., Vol. 17, pp. 1642-655, 1998) that overexpresses a defective form of the intracellular domain of the fibroblast growth factor (FGF) receptor (FGFR), which causes defects in multiple organs such as the kidney and lung, can also be used. Alternatively, nude mice can be used for hair or thymus production.

[0049] In the present invention, the non-human animal from which the recipient embryo is derived may be any animal other than a human, such as a pig, rat, mouse, cow, sheep, goat, horse, dog, chimpanzee, gorilla, orangutan, monkey, marmoset, bonobo, etc. It is preferable to collect embryos from a non-human animal whose adult size is similar to that of the animal species from which the organ is to be produced.

[0050] On the other hand, the mammal from which the cells to be transplanted into the recipient blastocyst stage fertilized egg to form the organ to be produced may be any mammal, whether human or non-human, such as pig, rat, mouse, cow, sheep, goat, horse, dog, chimpanzee, gorilla, orangutan, monkey, marmoset, bonobo, etc.

[0051] The relationship between the recipient embryo and the transplanted cells may be allogeneic or xenogeneic.

[0052] The cells to be transplanted prepared as described above are transplanted into the cavity of a recipient blastocyst-stage fertilized egg, and a chimeric cell mixture consisting of blastocyst-derived internal cells and the cells to be transplanted can be formed in the cavity of the blastocyst-stage fertilized egg.

[0053] The blastocyst-stage fertilized egg into which the cells have been transplanted is then implanted into the uterus of a pseudopregnant or pregnant female animal of the species from which the blastocyst-stage fertilized egg served as a foster mother. The blastocyst-stage fertilized egg is allowed to develop in the foster mother uterus to produce offspring. From the offspring, the desired organ can then be obtained as a mammalian cell-derived organ.

[0054] These and other advantages of the present invention will therefore become apparent from a reading of the following detailed description. [Effects of the Invention]

[0055] This invention provides an organ regeneration technology suitable for industrial use, enabling individuals to regenerate their own organs from somatic cells such as skin cells, depending on their individual characteristics.

[0056] Furthermore, by producing induced pluripotent stem cells (iPS cells) based on cells with a desired genome and practicing the present invention, it has become possible to conduct research and development using organs derived from various genomes, a technology that was completely impossible with conventional technology.

[0057] Furthermore, the use of iPS cells has the advantage that some of the ethical issues that have arisen with ES cells can be avoided, while still achieving the same effects. [Brief explanation of the drawings]

[0058] [Figure 1] This shows a therapeutic model using pancreatic construction derived from iPS cells by blastocyst complementation. [Figure 2](a) shows the strategy for establishing iPS cells from GFP mice. After establishing fibroblasts (Tail Tip Fibroblasts: TTFs) derived from the tails of GFP mice, three factors (reprogramming factors) were introduced and the cells were cultured in ES cell medium for 25–30 days. iPS colonies were picked and iPS cell lines were established. (b) shows the morphology of the established iPS cells, photographed with a camera microscope. GFP-iPS cell #2 is shown on the left, and #3 is shown on the right. (c) shows the measurement of alkaline phosphatase activity. iPS cells were photographed under a fluorescent microscope and stained with an alkaline phosphatase staining kit (Vector, Cat. No. SK-5200). From the left, a bright-field image, a GFP fluorescent image, and alkaline phosphatase staining are shown. (d) shows the identification of the three introduced factors (reprogramming factors) by PCR using genomic DNA. Genomic DNA was extracted from iPS cells and PCR was performed. From top to bottom, the expression of the Klf4, Sox2, Oct3 / 4, c-Myc, and Myog genes is shown. From left to right, GFP-iPS cells #2 and #3, Nanog-iPS (four-factor), and ES cells (NC) are shown as a control. The results for distilled water are shown on the far right. The insertion of the three factors into the iPS cells used in this invention was confirmed. e. RT-PCR analysis of the gene expression pattern characteristic of ES cells in the cells used in this invention and confirmation of the expression of the introduced genes are shown. From top to bottom, the expression patterns are Klf4, Sox2, Oct3 / 4, and c-Myc. The expression of the Nanog, Rex1, and Gapdh genes is shown. The negative control (RT(-)) is shown at the bottom. For Klf4, Sox2, and Oct3 / 4, the expression was confirmed separately for total RNA and transgenic (Tg) cells. From left to right, the expression patterns of GFP-iPS cells #2 and #3, ES cells (NC) as a control, and TTF (negative control) as a control are shown. The rightmost image shows the results for distilled water. f. shows the production of chimeric mice using iPS cells. The established iPS cells were injected into blastocysts obtained by mating C57BL6 and BDF1 mice, resulting in the production of chimeric mice. The top image shows a bright-field image (left) and a GFP fluorescent image (right) at embryonic day 13.5.The bottom shows the neonatal period. NC indicates the negative control. [Figure 3] Figure 3 shows the morphology of the pancreas constructed by blastocyst complementation (day 5 after birth). In the homo mouse, the pancreas edge is neatly composed of GFP-positive cells, while in the hetero mouse pancreas, it is a dot-like chimera. [Figure 4] Figure 4 shows histological analysis of iPS cell-derived pancreas (day 5 after birth). Frozen sections of iPS cell-derived pancreas were prepared and stained with DAPI as a nuclear stain, anti-GFP antibody, and anti-insulin antibody. Images were then observed and photographed using an upright fluorescence microscope and a confocal laser scanning microscope. From the left, a bright-field image, a GFP+DAPI image, and an anti-insulin antibody staining image are shown. The upper panel shows the Pdx1LacZ / LacZ clone of the present invention transfected with GFP-iPS cells, while the lower panel shows the control Pdx1wt / LacZ clone transfected with GFP-iPS cells. [Figure 5]This section shows an experiment confirming the existence of GFP-negative cells due to gene silencing. Bone marrow cells were collected from the same mouse shown in Figure 3, and GFP-hematopoietic stem / progenitor cells (c-Kit+, Sca-1+, Lineage marker-: KSL cells) were sorted using a flow cytometer and individually placed into a 96-well plate. These were cultured for 12 days under cytokine-containing conditions to form colonies, from which genomic DNA was extracted and used for genotyping. This allows for clonal gene determination from a single cell, even if the GFP-negative cells contain cells that have lost GFP expression due to gene silencing. This allows for easy differentiation of host cells from gene-silenced cells. (a) shows the colony formation strategy using KSL cells purified from bone marrow cells; (b) shows the morphology of blood colonies on day 12 of culture; and (c) shows the genotyping of chimeric individuals using DNA extracted from each colony. Panel a shows, from left to right, the FACS patterns of c-Kit+, Sca-1+, and Linage- (KSL) hematopoietic stem and progenitor cells in the bone marrow. Photographs in b show colonies on day 12 of culture, from left to right, a bright-field image in the center, and a GFP fluorescent image on the left. Panel c shows the results of genotyping DNA extracted from single-cell-derived colonies using the Qiagen kit described above, followed by PCR. PCR was performed using the same primers and conditions as used to determine Pdx1 offspring. [Figure 5A]Transplantation of iPS-derived islets into STZ-induced diabetic mice. Panels a and b show islet isolation. The iPS-derived pancreas was perfused with collagenase from the common bile duct (arrow in a), and after density gradient centrifugation, EGFP-expressing iPS-derived islets were enriched (b). Panel c shows the kidney capsule 2 months after islet transplantation. The EGFP-expressing spots (arrow) represent transplanted islets. Panel d shows HE staining of kidney sections (left panel) and GFP staining with DAPI (right panel). Panel e shows the transplantation of 150 iPS-derived islets into STZ-induced diabetic mice. The arrow indicates the time point at which an antibody cocktail (anti-IFN-γ, anti-TNF-α, anti-IL-1β) was administered. Intraperitoneal blood glucose levels were measured every week for up to 2 months after transplantation. STZ-induced diabetic mice transplanted with iPS islets are represented by black triangles (n=6), and STZ-induced diabetic mice not transplanted with iPS islets are represented by black squares (f) shows the glucose tolerance test (GTT) 2 months after islet transplantation. [Figure 6] This shows kidney regeneration by blastocyst complementation in a Sall1 knockout mouse. The results of genotyping of the Sall1 allele are shown at the top. It can be seen that mouse #3 was a Sall1 homozygous KO mouse. The bottom shows the morphology of the kidney (1 day after birth) regenerated by blastocyst complementation with iPS cells using mouse #3 as a host. It can be seen that in the homozygous KO mouse, the entire kidney is neatly composed of GFP-positive cells. It has been demonstrated that it is possible to create iPS cell-derived kidneys using Sall1 knockout mice. [Figure 7] The photographs show that blastocyst complementation was performed using B6-derived iPS cells, and the resulting chimeric mice were confirmed to have hair. #1 is a C57BL / 6 (B6) wild-type (control) mouse, and black hair can be seen. #3 is a KSN nude mouse (control), and is hairless. #2, #4, and #5 show the three resulting chimeric mice, which have grown hair. [Figure 8]This figure confirms the development of the thymus in chimeric and control mice. A thymus can be seen in C57BL / 6 (B6) wild-type mice (controls). A thymus is absent in nude mice. In contrast, a thymus can be seen in chimeric mice. [Figure 9] The results of fractionating peripheral blood from C57BL / 6 (B6) wild-type (control) mice and each of the chimeric mice (#2, 4, and 5) in Figure 7 into CD4 and CD8 positive cells (T cells) and analyzing GFP positive cells are shown. The degree of chimerism is indicated by the distribution of GFP negative and GFP positive cells. [Figure 10] Male Pdx1(- / -) mice (founder: Pdx1(- / -) mice whose pancreases were supplemented with mouse iPS cells) were mated with female Pdx1(+ / -) mice to obtain fertilized eggs. These were then developed in vitro to the blastocyst stage, and 10 EGFP-marked rat iPS cells were microinjected into the resulting blastocysts. These were then implanted into pseudopregnant foster mothers, and laparotomy was performed at full term. The resulting newborns were analyzed. Observation of EGFP fluorescence under a fluorescent stereomicroscope revealed EGFP expression on the body surface, revealing that individual numbers 1, 2, and 3 were chimeric. Laparotomy revealed pancreatic expression uniformly in #1 and #2. In contrast, the pancreas of #3 showed partial EGFP expression, but in a mosaic pattern. Furthermore, #4 is a littermate of #1-3, but lacks EGFP fluorescence on the body surface and is a non-chimeric Pdx1(- / -) mouse, as evidenced by the absence of a pancreas upon laparotomy. Spleens were also removed from these newborns, and blood cells extracted from them were stained with mouse or rat CD45 monoclonal antibodies and analyzed by flow cytometry. As a result, rat CD45-positive cells were observed alongside mouse CD45-positive cells in #1-3, confirming that these individuals are heterogeneous mouse-rat chimeras, consisting of a mixture of cells derived from host mouse and rat iPS cells. Furthermore, almost all cells in the rat CD45-positive cell fraction exhibited EGFP fluorescence, indicating that the rat CD45-positive cells were derived from rat iPS cells marked with EGFP. [Figure 10A]The Pdx1 genotypes of host mice #1 to #3 were confirmed by PCR. To confirm the genotypes of the host mice, the mouse CD45-positive cells shown in the dotted box in Figure 10 were collected from the same spleen samples as in Figure 10. Genomic DNA was extracted from these cells and subjected to PCR using primers capable of distinguishing between mutant and wild-type Pdx1 alleles. As a result, only mutant bands were observed in #1 and #2, while both mutant and wild-type bands were detected in #3. This indicates that the host genotypes were Pdx1(- / -) in #1 and #2, and Pdx1(+ / -) in #3. These results suggest that rat pancreases were successfully constructed within Pdx1(- / -) mice #1 and #2, which are not capable of developing a pancreas, by applying xenogeneic blastocyst complementation using rat iPS cells as donors. DETAILED DESCRIPTION OF THE INVENTION

[0059] The present invention will be described below. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the relevant field unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In case of conflict, the present specification (including definitions) will prevail.

[0060] For the purpose of specifically describing the embodiments of the present invention, exemplary embodiments are described below. As an example, a method for producing a kidney derived from mammalian cells in a mouse body is described below. It is understood that the pancreas, hair, and thymus can also be produced by such a method.

[0061] (non-human animals) To produce kidneys derived from non-human mammalian cells in vivo in an animal such as a mouse, an animal such as a mouse with an abnormality that results in the failure of kidney development during development is prepared. In one embodiment of the present invention, a Sall1 knockout mouse (Nishinakamura, R. et al., Development, Vol. 128, pp. 3105-3115, 2001) can be used as the mouse with an abnormality that results in the failure of kidney development during development. This animal, when homozygous for Sall1(- / -), is characterized by the failure of kidney development alone, resulting in the absence of kidneys in offspring. Alternatively, the founder animals described herein can also be used.

[0062] These mice are maintained in a heterozygous state (Sall1(+ / -)) for the Sall1 gene deficiency because kidneys do not form and the mice cannot survive if the Sall1 gene deficiency is homozygous (Sall1(- / -)). Mice with such heterozygous states are mated (Sall1(+ / -) x Sall1(+ / -)), and fertilized eggs are collected from the uterus. Fertilized eggs are stochastically generated with a Sall1(+ / +):Sall1(+ / -):Sall1(- / -) ratio of 1:2:1. In the present invention, Sall1(- / -) embryos, which occur with a 25% probability, are used. However, determining the genotype at the early embryonic stage is difficult. Therefore, it is more practical to determine the genotype of the offspring after birth and use only those individuals with the desired Sall1(- / -) genotype in subsequent processes.

[0063] In the creation of these knockout mice, the Sall1 gene may be knocked out and a gene for a detectable fluorescent protein, green fluorescent protein (GFP), may be knocked into the Sall1 gene region in an expressible state (Takasato, M. et al., Mechanisms of Development, Vol. 121, pp. 547-557, 2004). By knocking in such a fluorescent protein, activation of the regulatory region of this gene results in expression of GFP in place of Sall1, allowing the deficiency of the Sall1 gene to be determined by fluorescence detection.

[0064] In the present invention, the relationship between the recipient embryo and the transplanted cells may be either homospecific or heterospecific. Numerous reports have been published in the art on the creation of heterospecific chimeric animals, including those between closely related animal species, such as rat-mouse chimeras (Mulnard, JG, CR Acad. Sci. Paris. 276, 379-381 (1973); Stern, MS, Nature. 243, 472-473 (1973); Tachi, S. & Tachi, C. Dev. Biol. 80, 18-27 (1980); Zeilmarker, G., Nature. 242, 115-116 (1973)) and sheep-goat chimeras (Fehilly, C.B. et al., Nature. 307, 634-636 (1984)). Therefore, in the present invention, when, for example, a kidney derived from non-human mammalian cells is produced in vivo in a mouse, a heterologous organ can be produced in a recipient embryo based on these conventionally known chimera production methods (for example, a method in which cells to be transplanted are inserted into a recipient blastocyst (Fehilly, CB, et al., Nature, 307, 634-636 (1984))).

[0065] As used herein, the term "non-human mammal" refers to a mammal from which a chimeric animal or chimeric embryo, etc. is produced using transplanted cells.

[0066] As used herein, the term "allogeneic mammal" refers to any mammal that is an individual different from the above-mentioned non-human mammal, and may be an allogeneic or heterogeneous mammal.

[0067] As used herein, the term "non-human foster parent mammal" refers to a mammal in whose womb a fertilized egg, produced by transplanting cells derived from an allogeneic mammal that is different from the non-human mammal, develops (becomes a foster parent).

[0068] It should be understood that, although "non-human mammals" and "non-human foster mammals" are sometimes referred to as "non-human host mammals" or "hosts," "non-human mammals" and "non-human foster mammals" are different animals, and it would be clear to one skilled in the art which is being referred to in the context of the present invention.

[0069] When producing a pancreas as an organ, the recipient non-human embryo can be a Pdx-1 knockout animal (Offield, MF, et al., Development, Vol. 122, p. 983-995, 1996) that has an abnormality that prevents pancreas development during development, or an embryo of a founder animal described in this specification.

[0070] When producing hair as an organ, a nude mouse embryo, which does not grow hair, can be used as the recipient non-human embryo.

[0071] When producing a thymus as an organ, a nude mouse embryo can be used as the recipient non-human embryo.

[0072] (Cells to be transplanted) Next, to explain the cells to be transplanted, using the kidney as an example, iPS cells (see Non-Patent Document 2, etc.) are prepared as cells to be transplanted to produce a kidney derived from mammalian cells. These cells have a wild-type genotype for the Sall1 gene (Sall1(+ / +)) and have the ability to develop into all cells of the kidney.

[0073] Before transplantation, these cells may be incorporated with a fluorescent protein in an expressible state for specific detection. For example, a DsRed gene variant, DsRed.T4 (Bevis BJ and Glick BS, Nature Biotechnology Vol. 20, pp. 83-87, 2002), may be sequenced as such a fluorescent protein for detection so that it is expressed in almost all organs under the control of a CAG promoter (cytomegalovirus enhancer and chicken actin gene promoter), and then incorporated into iPS cells by electroporation. Such fluorescent proteins may be those known in the art, such as green fluorescent protein (GFP). By fluorescently labeling the cells for transplantation, it is possible to easily determine whether the resulting organ is composed solely of the transplanted cells.

[0074] These mouse iPS cells are transplanted into the lumen of a blastocyst-stage fertilized egg with the aforementioned Sall1(- / -) genotype to produce a blastocyst-stage fertilized egg with a chimeric inner cell mass, and these blastocyst-stage fertilized eggs with a chimeric inner cell mass are then developed in the uterus of a foster mother to produce offspring. If unmarked iPS cells are used for chimera production, there is no way to distinguish them from the host embryo, making it impossible to determine whether organ replenishment has occurred. Therefore, to solve this problem, fluorescent dyes can be introduced into iPS cell lines, and experiments can be performed using standard methods described in the Examples, etc.

[0075] (Method of producing founder animals for breeding) The breeding founder animals used in the present invention have the following characteristics: they contain a gene encoding the cause of the deficiency of an organ or body part that, if functional, would render the organ non-viable or difficult to survive, and the organ or body part is complemented by blastocyst complementation. By using this animal (also referred to as a "founder animal" herein) to produce subsequent generation animals, it is possible to produce an organ with the desired genomic type by causing the deficiency of the desired organ. Furthermore, it has been shown that organs can be produced in subsequent generations using this method, and that it can also be used with iPS cells, opening up the door to industrial applications of the present invention.

[0076] As used herein, the phrase "an organ or body part that would be non-viable or difficult to survive if it were to function" refers to an organ or body part that, when it is caused by a certain factor, becomes non-viable or difficult to survive if that factor causes the organ or body part to become defective or malfunction (e.g., abnormal). For example, in the case of a foreign gene, when that gene is introduced into an organism and is expressed normally, a defect occurs in a certain organ or body part, resulting in the organ or body part becoming non-viable or difficult to survive. Difficulty in survival includes the inability to produce offspring for the next generation and, in the case of humans, the resulting difficulty in social life. Examples of organs or body parts include, but are not limited to, the pancreas, liver, hair, and thymus.

[0077] Genes associated with such events include, for example, Pdx-1 (pancreatic counterpart).

[0078] For organ production, a gene must be selected that complements the organ and does not cause postnatal death due to other factors (such as the inability to obtain milk from the mother). An example of such a gene is Pdx-1. The present invention can be implemented using a gene with such properties. For example, even if the phenotype is similar to that of pancreatic deficiency, the implications are significantly different. Specifically, knockouts improve production efficiency, while transgenics also enable clonal analysis of lethal phenotypes.

[0079] As used herein, the phrase "cannot survive or makes it difficult to survive when it functions" refers to the situation where, when a certain factor functions, the host animal is completely unable to survive and dies, or the host animal is able to survive but is subsequently rendered virtually unable to survive due to reasons such as difficulty in growth or reproduction, and can be understood using common knowledge in the relevant field.

[0080] As used herein, the term "organ" is used in the usual sense in the art and generally refers to organs that constitute the body of an animal.

[0081] As used herein, the term "body part" refers to any part of the body, including those not generally referred to as organs. For example, taking the kidney as an example, when a normal gene is present, a complete kidney is generated. However, if a certain gene is missing or abnormal, an organ like a kidney may be formed, but an abnormality or defect may occur in a part of the organ. The part where such an abnormality or defect occurs can be considered an example of this "body part." Since gene deficiencies or abnormalities do not necessarily correspond to each organ and often affect only a part of the organ, when considering the correspondence with genes, it may be better to consider the correspondence in terms of body parts, and such correspondence will also be taken into account in this specification.

[0082] As used herein, "blastocyst complementation" refers to a technique for complementing missing organs or body parts by utilizing the phenomenon in which injection of pluripotent cells, such as ES cells or iPS cells, into the cavity of a blastocyst-stage fertilized egg results in the formation of a chimeric mouse. The present inventors have discovered that blastocyst complementation, which was previously thought to be difficult, can produce mammalian organs with complex cellular structures consisting of multiple cell types, such as kidney, pancreas, hair, and thymus, in living animals, particularly non-human animals, and have confirmed that this can also be achieved using iPS cells. Therefore, this technique can be fully utilized in the present invention using iPS cells.

[0083] As used herein, the term "label" refers to any factor that can be used to identify the complemented organ. For example, by expressing a specific gene (e.g., a gene expressing a fluorescent protein) only in the organ to be complemented, the organ to be complemented can be distinguished from the complemented host by the property (e.g., fluorescence) attributable to that specific gene. In this way, it is possible to distinguish whether the complementation was carried out using cells derived from external cells or internal cells, thereby making it easier to select founder animals for use in the present invention. Before transplantation, these cells may be incorporated in an expressible state with a fluorescent protein for specific detection. For example, the DsRed gene variant, DsRed.T4 (Bevis BJ and Glick BS, Nature Biotechnology Vol. 20, pp. 83-87, 2002), can be sequenced to be expressed in almost all organs under the control of the CAG promoter (cytomegalovirus enhancer and chicken actin gene promoter), and then incorporated into ES cells by electroporation. By labeling such transplant cells with fluorescence, it is possible to easily detect whether the produced organ is composed only of the transplanted cells.

[0084] Examples of such labels include green fluorescent protein (GFP) gene, red fluorescent protein (RFP), blue fluorescent protein (CFP), other fluorescent proteins, and LacZ.

[0085] The method for producing founder animals used in the present invention comprises the following steps: A) providing a first pluripotent cell carrying the gene; B) growing the first pluripotent cell into a blastocyst; C) introducing a second pluripotent cell capable of complementing the genetic defect into the blastocyst to produce a chimeric blastocyst; D) producing an individual from the chimeric blastocyst and selecting one in which the organ or part thereof has been complemented by the second pluripotent cell.

[0086] As used herein, the terms "a (deficiency-causing) gene encoding a deficiency factor that causes an organ or body part to become non-viable or to have difficulty surviving when it functions" or "deficiency-causing gene" are used interchangeably, and when referring to a gene, refer to a gene that causes an organ or body part to become non-viable or to have difficulty surviving when its function (for example, when it is introduced and expressed in the case of an exogenous gene, or when it is exposed to conditions in which such a gene functions in the case of an endogenous gene) causes the organ or body part to become non-viable or to have difficulty surviving.

[0087] As used herein, the term "pluripotent cells" includes egg cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), pluripotent germ stem cells (mGS cells), and the like.

[0088] As used herein, the term "first pluripotent cell" refers to a pluripotent cell or a cell mass derived therefrom that is used as the source of a host (also referred to as a host herein) such as a founder animal, and preferably a fertilized egg or embryo is used.

[0089] As used herein, the term "second pluripotent cells" refers to pluripotent cells used for the purpose of producing an organ, and iPS cells are used.

[0090] As used herein, the phrase "having the ability to complement a defect" refers to the ability to complement an organ or body part when referring to a factor, gene, or the like.

[0091] As used herein, the term "chimeric blastocyst" refers to a blastocyst formed by a chimeric state between a cell derived from a first pluripotent cell and a cell derived from a second pluripotent cell. Such chimeric blastocysts can be produced by injection methods, as well as by methods such as the "aggregation method," in which embryos are brought into close contact with each other in a petri dish to produce chimeric blastocysts. Furthermore, in the present invention, the relationship between the recipient embryo and the transplanted cells may be either homotypic or heterotypic. There have been many reports in the technical field on the creation of such chimeric animals between different species. For example, chimeric animals between closely related animal species have been reported, such as the creation of chimeras between rats and mice (Mulnard, JG, CR Acad. Sci. Paris. 276, 379-381 (1973); Stern, MS, Nature. 243, 472-473 (1973); Tachi, S. & Tachi, C. Dev. Biol. 80, 18-27 (1980); Zeilmarker, G., Nature, 242, 115-116 (1973)) and the creation of chimeras between sheep and goats (Fehilly, CB, et al., Nature, 307, 634-636 (1984)). Therefore, in the present invention, when, for example, a kidney derived from non-human mammalian cells is produced in vivo in a mouse, a heterologous organ can be produced in a recipient embryo based on these conventionally known chimera production methods (for example, a method in which cells to be transplanted are inserted into a recipient blastocyst (Fehilly, CB, et al., Nature, 307, 634-636 (1984))).

[0092] In the method for producing founder animals used in the present invention, the step of providing first pluripotent cells carrying a gene (also referred to herein as a "deficiency-causing gene") encoding a cause of a defect in an organ or body part that, if functional, would render the animal non-viable or difficult to survive can be carried out, for example, by procuring pluripotent cells carrying the gene, or by introducing the gene into pluripotent cells to produce pluripotent cells carrying the gene. Methods for introducing such genes are well known in the art, and those skilled in the art can select an appropriate method to carry out such gene introduction. Preferably, electroporation is used. This is because, by applying an electric pulse to a cell suspension, tiny holes are created in the cell membrane, allowing DNA to be delivered into the cells, thereby causing transformation (i.e., introduction of the gene of interest), resulting in less subsequent damage, but this is not limited to the above.

[0093] In the method for producing founder animals used in the present invention, the step of growing a first pluripotent cell (e.g., a fertilized egg, an embryo, etc.) into a blastocyst can be carried out by any known growth method for growing a pluripotent cell into a blastocyst. Such conditions are well known in the art and are described in "Manipulating the Mouse Embryo: A Laboratory Manual, 3rd Edition 2002" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is incorporated herein by reference.

[0094] In the method for producing founder animals used in the present invention, the step of introducing induced pluripotent stem cells (iPS cells), which are second pluripotent cells capable of complementing the genetic defect, into blastocysts to produce chimeric blastocysts may be performed by any method known in the art, as long as it is possible to introduce the second pluripotent cells, iPS cells, into blastocysts. Such methods include, but are not limited to, injection and aggregation.

[0095] In the method for producing founder animals used in the present invention, methods for producing individuals from chimeric blastocysts can be known in the art. Typically, the chimeric blastocyst is transferred to a foster mother, where it is allowed to become pregnant and grow in the uterus of the foster mother, but the method is not limited to this.

[0096] In the method for producing founder animals used in the present invention, the selection of animals with a complement of organs or body parts thereof can be carried out using any technique that can confirm the complement of the organs or body parts.

[0097] An example of such a method is the identification of an identifier derived from induced pluripotent stem cells (iPS cells), which are second pluripotent cells. As used herein, "identifier" refers to any factor capable of identifying an individual, species, etc., and identifying its origin, and is also abbreviated as "ID." Such an identifier may be, for example, a genomic sequence, phenotype, or the like, unique to the second pluripotent cells, iPS cells. Alternatively, such selection can be performed by using labeled or labelable second pluripotent cells (including those that become labeled through gene expression) and identifying the marker in the selection method for producing founder mice of the present invention. It will be understood that those skilled in the art can also improve and implement this method as appropriate.

[0098] (Organ regeneration method using founder animals) In another aspect, the present invention provides a method for producing a desired organ or body part using induced pluripotent stem cells (iPS cells) with a founder animal. This method comprises the steps of: A) providing a founder animal, in which the defect-causing gene in the founder animal encodes the cause of the defect in the desired organ or body part; B) obtaining eggs from the founder animal and growing them into blastocysts; C) introducing into the blastocysts induced pluripotent stem cells (iPS cells), which are desired pluripotent cells having a desired genome capable of complementing the genetic defect, to produce chimeric blastocysts; and D) producing an individual from the chimeric blastocyst and obtaining the desired organ or body part from the individual.

[0099] Here, this step D) can be carried out by developing the chimeric blastocyst in the womb of a non-human foster mammal to obtain a baby, and then obtaining the target organ from the baby.

[0100] (Pancreas formation) Pancreatic formation can be investigated by macroscopic or microscopic morphological analysis, gene expression analysis, etc. using methods such as macroscopic observation, microscopic observation after staining, or observation using fluorescence.

[0101] For example, macroscopic observations can be used to determine whether or not an organ actually exists and to examine its external appearance and other characteristics. In addition to this macroscopic morphological analysis, microscopic observations can also be performed using a microscope after staining with standard tissue stains such as hematoxylin-eosin staining. These microscopic observations allow for detailed investigations of the various cellular structures within the pancreas.

[0102] Furthermore, gene expression analysis using fluorescence can be performed by activating fluorescent dyes under specific conditions. For example, in the case of the Pdx1-Lac-Z knockout mice described above, wild-type (+ / +) or heterozygous (+ / -) individuals exhibit patchy chimeric fluorescence even when fluorescently labeled ES cells are used, whereas homozygous (- / -) individuals exhibit uniform fluorescence because the pancreas is constructed entirely from ES cell-derived cells. This characteristic can be utilized to easily determine the genotype of the Pdx1 gene in a target organ or the cells that comprise the organ. If unmarked iPS cells are used to generate chimeras, there is no way to distinguish them from the host embryo, making it impossible to determine whether the organ has been replenished. To address this issue, fluorescent dyes can be introduced into iPS cell lines, allowing experiments to be performed using a similar protocol. Using these cells, organs can be generated using a similar protocol to those used with iPS cells, and their origins can be elucidated.

[0103] (Kidney formation) Kidney formation can be investigated by macroscopic or microscopic morphological analysis, gene expression analysis, etc. using methods such as macroscopic observation, microscopic observation after staining, or fluorescence observation.

[0104] For example, macroscopic observations can be used to determine whether organs are actually present and their external appearance. In addition to this macroscopic morphological analysis, tissues can also be observed microscopically using a microscope after standard histological staining, such as hematoxylin-eosin staining. Such microscopic observations can also be used to investigate the specific cellular makeup of the kidney.

[0105] Furthermore, gene expression analysis using fluorescence can be performed by activating fluorescence depending on the conditions. For example, in the case of the Sall1 gene knockout mouse mentioned above, when the Sall1 gene is homozygous (Sall1(- / -)), GFP fluorescence is emitted from both alleles, resulting in less fluorescence than when the Sall1 gene is heterozygous (Sall1(+ / -)), in which fluorescence is emitted from only one allele. This characteristic can be utilized to easily determine the genotype of the Sall1 gene in a target organ or the cells that make up the organ. If unmarked iPS cells are used to generate chimeras, there is no way to distinguish them from the host embryo, making it impossible to determine whether the organ has been replenished. Therefore, to address this issue, introducing fluorescent dyes into iPS cell lines can reveal their origin.

[0106] (hair formation) Hair formation can be investigated by macroscopic observation, or by macro- or micro-morphological analysis using methods such as fluorescence observation, gene expression analysis, and the like.

[0107] For example, by performing macroscopic observations, it is possible to examine whether hair actually exists and its characteristics, such as its appearance. In addition to such macroscopic morphological analysis, it is also possible to observe tissues microscopically using a microscope after general tissue staining such as hematoxylin-eosin staining. Such microscopic observations allow for the investigation of the various cellular structures within specific hairs.

[0108] Furthermore, gene expression analysis using fluorescence can be performed by emitting fluorescence under appropriate conditions. For example, in the case of nude mice, the strong autofluorescence of hair makes it extremely difficult to visually determine whether the resulting hair is nude mouse-derived or iPS cell-derived under a fluorescence microscope. However, this can be achieved by appropriate fluorescent observation techniques. Taking advantage of this characteristic, the genotype of a target organ or the cells that make up the organ can be easily determined. If unmarked iPS cells are used for chimera production, there is no way to distinguish them from the host embryo, making it impossible to determine whether the organ has been replenished. Therefore, to address this issue, fluorescent dyes can be introduced into iPS cell lines, allowing experiments to be performed using a similar protocol. Using these cells, organs can be created using a similar protocol to that used with iPS cells, and their origins can be clarified.

[0109] (Thymus formation) Thymus formation can be investigated by macroscopic or microscopic morphological analysis, gene expression analysis, etc. using methods such as macroscopic observation, microscopic photography, FACS, or fluorescence observation.

[0110] For example, macroscopic observations can be used to examine whether an organ is actually present and its external appearance. In addition to such macroscopic morphological analysis, microscopic observations can also be performed using a microscope after standard histological staining, such as hematoxylin-eosin staining. Such microscopic observations can also be used to examine the specific cellular composition within the thymus.

[0111] Furthermore, gene expression analysis using fluorescence can be performed by emitting fluorescence under certain conditions. For example, the nude mouse mentioned above does not traditionally have a thymus, but this does not affect survival, and the mouse is born naturally with the thymus missing. When fluorescently labeled iPS cells are injected into these mice through blastocyst complementation, many of the individuals in which the iPS cells are confirmed to have contributed have a fluorescent thymus. Taking advantage of this characteristic, the genotype of the target organ or the cells that make up the organ can be easily determined.

[0112] (iPS cells) iPS cells can also be generated by other methods. Specifically, iPS cells can be produced by inducing reprogramming by contacting somatic cells with reprogramming factors (which may be a single factor or a combination of factors). Examples of such reprogramming and reprogramming factors include the following. For example, in the examples of the present invention, the inventors independently generated iPS cells using fibroblasts collected from the tails of GFP transgenic mice with three factors (Klf4, Sox2, and Oct3 / 4; these are representative "reprogramming factors" used in the present invention). However, other combinations, such as the four factors Oct3 / 4, Sox2, Klf4, and c-Myc (also known as Yamanaka factors), can also be used, and improved methods can also be used. iPS cells can also be established using n-Myc instead of c-Myc and a lentiviral vector, a type of retroviral vector (Blelloch R et al., (2007). Cell Stem Cell 1:245-247). In addition, human iPS cells have been successfully established by introducing four genes, Oct3 / 4, Sox2, Nanog, and Lin28, into fetal lung fibroblasts and neonatal foreskin fibroblasts (Yu J, et al., (2007). Science 318:1917-1920).

[0113] Human iPS cells can also be produced from fibroblast-like synoviocytes and neonatal foreskin-derived fibroblasts using Oct3 / 4, Sox2, Klf4, and c-Myc, which are human homologs of the mouse genes used to establish mouse iPS cells (Takahashi K, et al., (2007). Cell 131: 861-872). Human iPS cells can also be established using six genes: Oct3 / 4, Sox2, Klf4, and c-Myc, plus hTERT and SV40 large T (Park IH, et al., (2007). Nature 451:141-146). Furthermore, it has been shown that iPS cells can be established in mice and humans using only the three factors Oct-4, Sox2, and Klf4, albeit at a low efficiency, without introducing the c-Myc gene, and that this has been successful in suppressing the transformation of iPS cells into cancer cells, and therefore this can also be used in the present invention (Nakagawa M, et al., (2008). Nat Biotechnol 26:101-106.; Wering M, et al., (2008). Cell Stem Cell 2:10-12).

[0114] The target organ obtained by this invention is characterized by being completely derived from the heterologous mammal. Conventional methods have resulted in the regeneration of chimeras. Without wishing to be bound by theory, this is thought to be due to the missing gene's function during development, particularly that of a transcription factor essential for the differentiation and maintenance of stem / progenitor cells of each organ during organogenesis. iPS cells can be used. The production of iPS cells is as described above. However, since the iPS cell line known as Nanog-iPS is not marked, when used to generate chimeras, there is no way to distinguish it from the host embryo, making it impossible to determine whether the organ has been replenished. To address this issue, introducing a fluorescent dye into this Nanog-iPS cell line allows experiments to be performed using the same protocol as for ES cells. Using these cells, it is possible to create organs using the same protocol as for ES cells and clarify their origin.

[0115] The present invention also provides mammals produced by the methods of the present invention. Because animals with such target organs have not previously been produced, the animals themselves are considered to be valuable as inventions. Without wishing to be bound by theory, it is believed that the reason such animals have not previously been produced is that the missing organs caused by gene deficiencies are essential for survival, and there has been no method to rescue them.

[0116] The present invention also provides the use of a non-human mammal having an abnormality in which a target organ does not develop during development, for the production of a target organ. The use of host cells for such purposes has not previously been fully anticipated. Therefore, such animals themselves are considered to have value as inventions. Without wishing to be bound by theory, it is believed that the reason such animals have not been produced until now is that the defective organ, exhibited by the gene deficiency, is essential for survival, and it has been impossible to maintain the target individual until the age at which sexual maturity is reached.

[0117] (Points to note when using various animals) When using animals other than mice, the techniques described in the Examples of this specification can be applied by paying attention to the following points. For example, with regard to the production of chimeras in animals of other species, there have been more reports of chimeras obtained by injecting embryos or, within embryos, the inner cell mass that is the origin of ES cells (rat: (Mayer, JR. & Fretz, HI. The culture of preimplantation rat embryos and the production of allophenic rats. J. Reprod. Fertil. 39, 1-10 (1974)); cattle: (Brem, G. et al. Production of cattle chimeras through embryo microsurgery. Theriogenology. 23, 182 (1985)); pig: (Kashiwazaki N et al., Production of chimeric pigs by the blastocyst injection method. Vet. Rec. 130, 186-187 (1992))) than reports of the establishment of pluripotent stem cells capable of forming chimeras in species other than mice. However, the methods described herein can also be applied to chimeras obtained by injection of inner cell masses. As described above, it is practically possible to use the inner cell mass to replace missing organs in animals. That is, for example, any of the above cells can be cultured in vitro until they reach the blastocyst stage, and then a portion of the inner cell mass can be physically detached from the resulting blastocyst and injected into another blastocyst. Chimeric embryos can also be produced by aggregating intermediate 8-cell stage embryos or morulae.

[0118] (General technology) The molecular biological, biochemical, and microbiological techniques used herein are well known and commonly used in the art, and may be selected from the group consisting of: Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates;Ausubel,FM(1995).Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates;Innis,MAet al.(1995).PCR Strategies,Academic Press;Ausubel,FM(1999).These methods are described in "Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology," Wiley, and annual updates; Sninsky, JJ et al. (1999); "PCR Applications: Protocols for Functional Genomics," Academic Press; and "Experimental Methods for Gene Transfer and Expression Analysis," a special edition of Experimental Medicine, Yodosha, 1997, among others, and relevant portions (possibly in their entirety) of these methods are incorporated herein by reference.

[0119] DNA synthesis techniques and nucleic acid chemistry for producing artificially synthesized genes are described, for example, in Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. Let al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G. M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G. T. (1996). Bioconjugate Techniques, Academic Press, and the like, the relevant portions of which are incorporated herein by reference.

[0120] All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0121] The present invention has been described above by showing preferred embodiments for ease of understanding. The present invention will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims. [Example]

[0122] In this example, the following experiment was carried out in accordance with the spirit of animal welfare and in accordance with the standards for animal handling established by the University of Tokyo.

[0123] (Example of iPS cell preparation) We generated induced pluripotent stem (iPS) cells using fibroblasts harvested from the tails of GFP transgenic mice with three factors (Klf4, Sox2, and Oct3 / 4). The protocol is as follows. The scheme is shown in Figure 1 and in detail in Figure 2a.

[0124] (Establishment of GFP mouse tail-derived fibroblasts (Tail tip fibroblast: TTF)) Approximately 1 cm of tail was collected from a GFP transgenic mouse, skinned, and minced into 2-3 pieces. The pieces were placed in MF-start medium (TOYOBO, Japan) and cultured for 5 days. The fibroblasts that emerged were then plated onto a new culture dish and passaged several times. These were designated tail-derived fibroblasts (TTF).

[0125] (Introduction of +3 factors (initialization factors)) The supernatant was collected from a virus-producing cell line (293gp or 293GPG cell line) that had been prepared by introducing the target gene and viral envelope protein. The virus solution, which had been centrifuged and then frozen, was added to the culture medium of TTF cells that had been passaged the day before to a concentration of 1 x 105 cells per 6-well plate, and this was used to introduce the three factors (reprogramming factors).

[0126] (Culture in ES cell medium for 25-30 days) After the introduction of the three factors (reprogramming factors), the medium was replaced with ES culture medium the next day and cultured for 25 to 30 days. During this time, the medium was replaced every day.

[0127] (Picking up iPS colonies and establishing iPS cell lines) iPS cell-like colonies that emerged after culturing were picked up using a yellow tip (e.g., available from Watson), disaggregated into single cells with 0.25% trypsin / EDTA (Invitrogen), and plated onto freshly prepared mouse embryonic fibroblasts (MEFs).

[0128] (result) The iPS cell lines established by the above method were proven to have the characteristics of iPS cells, namely, undifferentiated state and totipotency, as shown in Figure 2b-f.

[0129] The results of the above experiment are shown in Figure 2. As shown in Figure 2b, the morphology of the two established iPS cell lines was photographed using a camera-equipped microscope under the following conditions:

[0130] After the iPS cells were picked up and passaged, they were observed and photographed when they became semi-confluent on the dish.

[0131] It was found that the cells formed morphologically undifferentiated colonies similar to those of ES cells.

[0132] As shown in Figure 2c, iPS cells were photographed under a fluorescent microscope and stained with an alkaline phosphatase staining kit (Vector, Cat. No. SK-5200) under the following conditions:

[0133] Bright-field and GFP fluorescent images were observed and photographed using a microscope equipped with a camera. After removing the culture medium and washing the iPS cell culture dish with phosphate-buffered saline (PBS), a fixative consisting of 10% formalin and 90% methanol was added and the dish was fixed for 1-2 minutes. After washing once with washing solution (0.1M Tris-HCl (pH 9.5)), the staining solution from the kit was added and the dish was left to stand in the dark for 15 minutes. After washing again with washing solution, the dish was observed and photographed.

[0134] As shown in Figure 2c, the iPS cells produced in this example were derived from GFP mice and therefore constitutively expressed GFP and exhibited high alkaline phosphatase activity, which is characteristic of undifferentiated cells.

[0135] As shown in Figure 2d, to identify the three factors inserted into the genomic DNA during iPS cell establishment, genomic DNA was extracted from the iPS cells and PCR was performed under the following conditions:

[0136] Genomic DNA was extracted from 1 x 10 cells using a DNA mini kit (Qiagen) according to the manufacturer's protocol. PCR was performed using the DNA as a template and the following primers: Oct3 / 4Fw (mOct3 / 4-S1120): CCC TGG GGA TGC TGT GAG CCA AGG (SEQ ID NO: 1) Rv(pMX / L3205): CCC TTT TTC TGG AGA CTA AAT AAA (SEQ ID NO: 2) Klf4Fw (Klf4-S1236): GCG AAC TCA CAC AGG CGA GAA ACC (SEQ ID NO: 3) Rv(pMXs-AS3200): TTA TCG TCG ACC ACT GTG CTG CTG (SEQ ID NO: 4) Sox2Fw (Sox2-S768): GGT TAC CTC TTC CTC CCA CTC CAG (SEQ ID NO: 5) Rv(pMX-AS3200): same as above (SEQ ID NO: 4) c-MycFW (c-Myc-S1093): CAG AGG AGG AAC GAG CTG AAG CGC (SEQ ID NO: 6) Rv(pMX-AS3200): same as above (SEQ ID NO: 4) As a result, as shown in FIG. 2d, insertion of the three factors was confirmed.

[0137] As shown in Figure 2e, the gene expression pattern characteristic of ES cells and the expression of the introduced genes were confirmed by reverse transcription polymerase chain reaction (RT-PCR) under the following conditions.

[0138] 1 x 105 GFP-positive cells were sorted into Trizol-LS Reagent (Invitrogen) using a flow cytometer, and cDNA was synthesized from the mRNA extracted from them using the ThermoScript RT-PCR System kit (Invitrogen) according to the attached protocol. PCR reactions were performed using the synthesized cDNA as a template. The primers used for transgene expression (denoted as Tg in the figure) were the same as those shown in Figure 2d above, and for other gene expression, primers synthesized based on the report by Takahashi K & Yamanaka S (Cell 2006 Aug 25;126(4):652-5.) were used.

[0139] As shown in Figure 2e, all lines showed expression patterns similar to those of ES cells, and it was found that the expression of the introduced gene (Tg) was suppressed due to the high gene silencing activity of iPS cells.

[0140] As shown in Figure 2f, the established iPS cells were injected into blastocysts to generate chimeric mice under the following conditions.

[0141] Oocytes collected from BDF1 mice (female, 8 weeks old) that had been superovulated with PMSG and hCG hormones were used for in vitro fertilization (IVF) with sperm from C57BL / 6 mice to obtain fertilized eggs. These were cultured to the 8-cell stage / morula, then cryopreserved and induced the day before blastocyst injection. Semiconfluent iPS cells were detached with 0.25% Trypsin / EDTA and suspended in ES cell culture medium for injection. Blastocyst injection was performed using a micromanipulator under a microscope, similar to the method used for blastocyst complementation. After incubation, the embryos were transferred to ICR surrogate uteruses. For analysis, embryos were observed and photographed under a fluorescent stereomicroscope on embryonic day 13 and postnatal day 1.

[0142] As shown in Figure 2f, iPS cell-derived cells (GFP positive) were confirmed at the fetal and neonatal stages, suggesting that the established iPS cell line has high pluripotency.

[0143] Example 1 In this example, mice were selected as founder animals, and the pancreas was selected as the organ to be deleted. Furthermore, the Pdx1 gene was used to generate knockout mice characterized by pancreatic deficiency.

[0144] (Mouse used) We used Pdx1wt / LacZ and Pdx1LacZ / LacZ (founder) knockout mice characterized by pancreatic deficiency. We also used blastocysts derived from mice in which the LacZ gene was knocked in (and knocked out) at the Pdx1 locus (Pdx1-LacZ knockin mice).

[0145] (Pdx1-LacZ knock-in mouse) Construct preparation can be carried out in detail based on a previously published paper (Development 122, 983-995 (1996)). Briefly, the procedure is as follows: Homologous region arms cloned from a λ clone containing the Pdx1 region can be used. In this example, those provided by Dr. Yoshiya Kawaguchi of the Laboratory of Surgical Oncology, Graduate School of Medicine, Kyoto University, were used.

[0146] (Transgenic knock-in technique: Pdx1-LacZ knock-in mouse) The constructs were electroporated into iPS cells prepared as described above, followed by positive / negative selection and screening by Southern blotting. The resulting clones were then injected into blastocysts to generate chimeric mice. Subsequently, germline-compatible lines were established, and the genetic background could be backcrossed to the C57BL / 6 strain to generate chimeric mice.

[0147] (Founder Mouse) (Mouse used) Transgenic mice characterized by pancreatic deficiency (Pdx1-Hes1 mice) were generated by injecting a construct containing the Hes1 gene under the Pdx1 promoter region into pronuclear-stage oocytes. The construct was generated by inserting the Hes1 gene (NCBI accession number NM_008235 mRNA) into the Pax6 gene region of a construct containing the Pdx1 promoter region, which was previously used in a paper (Diabetologia 43, 332-339 (2000)).

[0148] (Transgenic techniques) The above construct is injected using a microinjector into pronuclear stage eggs obtained by mating C57BL6 mice and BDF1 mice (purchased from Japan SLC Co., Ltd.), and the eggs are transplanted into foster mothers to produce transgenic mice.

[0149] The degree of pancreatic formation varies depending on the expression level of Hes1, which is expressed under the promoter of Pdx1 (particularly expressed in the fetal pancreas). High expression (i.e., high copy number) results in pancreatic deficiency. Pancreatic regeneration has been demonstrated by blastocyst complementation of Pdx1-Hes1 transgenic mice. It is possible to create a pancreas derived from iPS cells using these mice.

[0150] This demonstrates that the transgenic mice generated in this manner, like the Pdx1 knockout mice, are capable of pancreatic replacement.

[0151] (Generation of founder transgenic mice) Since the above transgenic mice are known to die after birth, founder mice of such mice are produced.

[0152] Briefly, embryos injected with the Pdx1-Hes1 transgene are cultured to the blastocyst stage, and then iPS cells are injected into them using a micromanipulator under a microscope to compensate for the pancreatic defect. The iPS cells used in this process are marked with GFP, as in the knockout section. Equivalently marked iPS cells can also be used. After injection, the embryos are transferred to the uterus of a surrogate mother, allowing for the production of offspring. This double embryonic manipulation, injecting iPS cells into transgene-introduced embryos, allows for pancreatic complementation in the first generation of transgenic animals, which can then serve as founder animals capable of transmitting the phenotype of pancreatic deficiency to subsequent generations.

[0153] (Mating) In this example, heterozygous mice were crossed with each other to obtain the knock-in mice. Because the knock-in mice described above cannot be maintained homozygously (they die within about one week after birth), we crossed Pdx1wt / LacZ and Pdx1LacZ / LacZ (founder) mice with each other and collected embryos.

[0154] (Mouse maintenance procedures and checks) The iPS cells were injected into the blastocysts under a microscope using a micromanipulator (Figure 1: Blastocyst injection of iPS cells). Conventional methods require marking with GFP, but in this case, iPS cells were established in advance from the somatic cells of GFP mice, so there was no need to mark them later and they were used as is. Of course, other iPS cells with equivalent markings may also be used. After injection, the embryos were transplanted into the uterus of a surrogate mother, and offspring were obtained.

[0155] Since the probability of the offspring being homozygous for knock-in mice is 1 / 4, it is necessary to determine which mice are the desired "pancreas-deficient + iPS cell-derived pancreas" mice. To do this, blood and tissue cells were collected from both groups, and GFP-negative cells (cells derived from the injected embryo, not from iPS cells) were sorted using a flow cytometer. Genomic DNA was extracted, and the genotype was detected by PCR to determine which mice were the winning mice. The primers used are as follows: Forward (Fw): ATT GAG ATG AGA ACC GGC ATG (SEQ ID NO: 7) Reverse 1 (Rv1): TTC AAC ATC ACT GCC AGC TCC (SEQ ID NO: 8) Reverse (Rv2): TGT GAG CGA GTA ACA ACC (SEQ ID NO: 9).

[0156] When produced using this method, because it is a heterozygous mating, the offspring are expected to follow Mendelian inheritance, with a wild-type: heterozygous: KO ratio of 1:2:1. Therefore, to identify the KO individuals, genotype analysis is performed using host-derived cells in the peripheral blood.

[0157] As a first step to confirm whether the replaced organ is functioning normally, we will analyze the expression of functional markers during the neonatal period, when pancreatic morphology can be easily observed.

[0158] This image shows an immunostained frozen section prepared from the pancreas of a mouse dissected at the neonatal stage. The section was stained using an anti-insulin antibody (purchased from Nichirei Biosciences, Inc., cat. #422421) as an indicator of endocrine tissue. Other antibodies that can be used to stain the sections include anti-α-amylase antibody (purchased from SIGMA, cat. #A8273), anti-glucagon antibody (purchased from Nichirei Biosciences, Inc., cat. #422271), and anti-somatostatin antibody (purchased from Nichirei Biosciences, Inc., cat. #422651) as indicators of exocrine tissue, and DBA-lectin (purchased from Vector, cat. #RL-1032) as an indicator of the pancreatic duct. The positive staining for insulin indicates that the complemented pancreas is functioning normally, even without staining with other antibodies.

[0159] These results indicate that almost all functional markers were expressed, suggesting that the cells have normal functions sufficient for survival.

[0160] The next step to confirm whether the replaced organs are functioning normally is to measure blood glucose levels in adult mice.

[0161] The results of pancreatic function evaluation in pancreatic complement mice using blood glucose levels as an indicator can be taken into consideration. The mean and standard deviation of steady-state blood glucose levels measured in mature pancreatic complement mice using a Medisafe Mini GR-102 (purchased from TERUMO) can be taken into consideration. Chimeric mice heterozygous for the Pdx1 allele and STZ-DM models with impaired pancreatic function can be used as controls. Changes in blood glucose levels after glucose loading, measured using the same Medisafe Mini, can also be taken into consideration. These results demonstrate normal blood glucose regulation and suggest that the generated pancreatic complement mice will be viable for long periods of time when used as founder mice.

[0162] In other words, after glucose loading, blood glucose levels that had risen once returned to normal, just like the heterozygous (+ / -) chimeras used as controls. Therefore, the KO chimeras (founders) created did not show symptoms such as diabetes, indicating the possibility of long-term survival.

[0163] Next, we attempted to determine whether the founder mice could transmit the phenotype to the next generation by mating them with heterozygous individuals. Genomic DNA was extracted from the tails of the resulting offspring and subjected to PCR using the primers used in the section (Mouse Maintenance Procedure and Verification). The results revealed that only heterozygous or knockout individuals were obtained, strongly suggesting that the founder mice were knockout individuals and could transmit the phenotype to the next generation.

[0164] In other words, because the knockout (KO) and heterozygous mice were crossed, theoretically, according to the laws of Mendelian inheritance, there should be a 1 / 2 probability of the resulting individual being KO or heterozygous, and this was the result shown.

[0165] This means that, for example, if two KO mice with complemented pancreases are bred together, it will be possible to obtain KO individuals in the next generation 100%, which will make it much easier to conduct analysis using KO individuals.

[0166] (Confirmation of Chimera) Chimeras can be identified by coat color. Because the donor iPS cells are derived from GFP transgenic mice and the host embryos are derived from wild-type C57BL6xBDF1 (black), GFP fluorescence can be used to identify chimeras. Transgenicity is determined by PCR of genomic DNA extracted from the tail to detect the transgene.

[0167] If the offspring are transgenic, the probability of the transgene being passed on to the next generation is 50%, so it is necessary to determine which mice have the desired "pancreatic deficiency + iPS cell-derived pancreas." To do this, we crossed them with wild-type mice, and confirmed the transmission of the transgene by detecting the genotype using PCR with genomic DNA extracted from the tails of the offspring, and also observed the morphology of the pancreas of the offspring. The following primer set was used for PCR: Forward (Fw): TGA CTT TCT GTG CTC AGA GG (SEQ ID NO: 10) Reverse (Rv): CAA TGA TGG CTC CAG GGT AA (SEQ ID NO: 11).

[0168] The forward primer used was designed to hybridize to the nucleotide sequence corresponding to the Pdx1 promoter region, and the reverse primer was designed to hybridize to the nucleotide sequence corresponding to Hes1 These primers were engineered to hybridize to the nucleotide sequence of the Pdx1 cDNA (mRNA with accession number NM_008235). Because the Pdx1 promoter and Hes1 cDNA do not exist in close proximity in wild-type mice, PCR using these primers allows for efficient detection of the transgene.

[0169] The above experiments suggest that the founder mice are capable of causing pancreatic deficiency in the next generation.

[0170] By applying this method not only to mice but also to other large animals, it should be possible to more efficiently produce transgenic and knockout animals with lethal phenotypes.

[0171] The transgenic chimeras created are then mated with wild-type mice. Morphological analysis of the offspring's pancreas or PCR of genomic DNA is used to determine whether the phenotype of pancreatic deficiency is transmitted to the next generation. If a transgenic chimera can serve as a founder, mice lacking a pancreas will be born in the next generation. Those mice successfully lacking a pancreas can then be selected. Because the pancreas was supplemented during the creation process, these mice will exhibit normal behavior after birth. In this way, even with transgenics, organ regeneration can be achieved using founders that can efficiently produce mice lacking organs, such as mice that die during the fetal stage or shortly after birth, along with iPS cells.

[0172] From the above, it has been demonstrated that mice containing those that have been rendered unable to produce a pancreas by forced expression of HES-1 using iPS cells, as well as Pdx-1 knockout mice, can be rescued from organ failure by scutellum complementation and used as founder mice.

[0173] (Pancreatic regeneration) Figure 3 shows pancreatic regeneration. Here, a 5-day-old newborn was dissected under a microscope, and the pancreas was exposed. It was then observed and photographed under a fluorescent microscope. The resulting photographs are shown in Figure 3.

[0174] (Morphology of iPS cell-derived pancreas) The morphology of the iPS cell-derived pancreas is shown in Figure 4. Frozen sections of the iPS cell-derived pancreas were prepared and stained with DAPI as a nuclear stain, as well as anti-GFP and anti-insulin antibodies, and then observed and photographed using an upright fluorescence microscope and a confocal laser scanning microscope.

[0175] From Figures 3 and 4, it appears that blastocyst complementation was achieved morphologically.

[0176] Figure 5 illustrates the method for determining the genotype of the host mouse. Bone marrow cells were collected from the same mouse shown in Figure 3, and GFP-negative hematopoietic stem / progenitor cells (c-Kit+, Sca-1+, Lineage marker-: KSL cells) were sorted using a flow cytometer and placed individually into a 96-well plate. These were cultured under cytokine-containing conditions for 12 days to form colonies, from which genomic DNA was extracted and used for genotyping. This method allows for clonal gene determination from a single cell, even if the GFP-negative cells contain cells that have lost GFP expression due to gene silencing. This allows for easy differentiation between host cells and gene-silenced cells. Note that the experiment in Figure 5 was intended to confirm the establishment of blastocyst complementation (whether this occurred due to organ vacancy (=knockout (KO))). To confirm the KO status by genotyping, single-cell genotyping was performed, taking into account the effects of gene silencing (Figure 5). a) shows the strategy, b) shows the image of colonies formed after cultivation, and c) shows the results of the evaluation.

[0177] (Consideration) As described above, organ regeneration was demonstrated using iPS cells independently generated from fibroblasts collected from the tails of GFP transgenic mice expressing three factors (Klf4, Sox2, and Oct3 / 4). Because Pdx1 knockout mice were crossed with a homozygous heterozygous mouse, there should have been a 50% chance of producing a homozygous pancreatic-deficient mouse, and this was demonstrated. Furthermore, based on the morphology of the iPS cell-derived pancreas shown in Figure 4 and the results of PCR analysis of isolated GFP-positive and GFP-negative cells shown in Figure 5, it can be said that there should have been a 50% chance of producing a homozygous pancreatic-deficient mouse, and this was demonstrated.

[0178] (Transplantation of iPS-derived pancreatic islets into STZ-induced diabetic mice) (Mouse used) C57BL / 6, BDF1, DBA2, and ICR mice were purchased from Japan SLC Co., Ltd. Pdx1 heterozygous (Pdx1(+ / -)) mice (provided by Dr. Yoshiya Kawaguchi, Graduate School of Medicine, Kyoto University, and Dr. Wright, Vanderbilt University) were crossed with DBA2 or BDF1 mice. C57BL / 6 mice were used as donors for the streptozotocin (STZ)-induced diabetes model. STZ (200 mg / kg) was administered intravenously after 16–20 h of fasting. Mice with blood glucose levels exceeding 400 mg / dL 1 week after STZ injection were considered hyperglycemic diabetic.

[0179] (mES / miPS cell culture) Undifferentiated mouse embryonic stem (mES) cells (G4.2) were maintained in a gelatin-coated dish without feeder cells in Glasgow's modified Eagle's medium (GMEM; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Nichirei Biosciences), 0.1 mM 2-mercaptoethanol (Invitrogen, San Diego, CA), 0.1 mM non-essential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 1% L-glutamine, penicillin, and streptomycin (Sigma), and 1000 U / ml leukemia inhibitory factor (LIF; Millipore, Bedford, MA). These G4.2 cells (kindly provided by Dr. Hitoshi Niwa of the RIKEN CDB) were derived from EB3 ES cells and harbored the enhanced green fluorescent protein (EGFP) gene under the control of the CAG expression unit. The ES cells are a sublineage derived from E14tg2a ES cells (Hooper M. et al., 1987) and were established by targeting the Oct-3 / 4 allele with the Oct-3 / 4-IRES-BSD-pA vector, which was constructed to express the drug resistance gene blasticidin under the control of the Oct-3 / 4 promoter (Niwa H. et al., 2000).

[0180] Undifferentiated mouse induced pluripotent stem (miPS) cells (GT3.2) were maintained on mitomycin-C-treated mouse embryonic fibroblasts (MEFs) in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 15% knockout serum replacement supplement (KSR; Invitrogen), 0.1 mM 2-mercaptoethanol (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), 1 mM HEPES buffer (Invitrogen), 1% L-glutamine, penicillin, and streptomycin (Sigma), and 1,000 U / ml leukemia inhibitory factor (LIF; Millipore). GT3.2 cells were established from tail fibroblasts of male GFP transgenic mice (kindly provided by Dr. Masaru Okabe, Osaka University) transfected with three reprogramming factors, Klf4, Sox2, and Oct3 / 4, via retroviral vectors. GT3.2 cells ubiquitously express EGFP under the control of the CAG expression unit.

[0181] Embryo culture and manipulation Pdx1 heterozygous (Pdx1(+ / -)) cross-bred embryos were prepared according to a previously published protocol (Nagy A. et al., 2003). Briefly, mouse 8-cell / morula-stage embryos were collected from the oviducts and uteri of Pdx1 heterozygous mice 2.5 days post-mating into M2 medium (Millipore). These embryos were transferred into drops of KSOM-AA medium (Millipore) and cultured for 24 hours to the blastocyst stage.

[0182] For embryo manipulation, blastocysts were transferred into microdroplets containing M2 medium, and mES / miPS cells were trypsinized and suspended in the microdroplets of culture medium. At the 8-cell / morula stage, embryos were transferred into microdroplets containing HEPES-buffered mES / miPS culture medium. After carefully piercing the zona pellucida and trophectoderm under a microscope using a piezo-driven micromanipulator (Primetech), 10–15 mES / miPS cells were injected into the blastocyst cavity near the inner cell mass (ICM). After injection, embryos were cultured in KSOM-AA medium for 1–2 hours and then transferred into the uterus of pseudopregnant, mated female ICR mice at 2.5 dpc.

[0183] (Isolation and transplantation of pancreatic islets) Pancreatic islets were isolated from mice bearing iPS-derived pancreases by collagenase digestion and then separated by centrifugation on a Ficoll gradient. Briefly, 10- to 12-week-old adult mice were sacrificed, and the pancreas was perfused with 2 mg / ml collagenase (Yakult) in Hank's balanced salt solution (HBSS; Invitrogen) via the bile duct using a 27G butterfly needle. The perfused pancreas was dissected and incubated at 37°C for 20 minutes. The digested fraction was washed twice with HBSS, and undigested tissue was removed using a strainer. The fraction was separated by density gradient centrifugation using Ficoll PM400 (GE-Healthcare, Stockholm, Sweden) in HBSS, and the islet-enriched fraction was collected in RPMI medium (Invitrogen) containing 10% FCS. Pancreatic islets with a diameter of approximately more than 150 μm were collected into a tube under a microscope using a glass micropipette.

[0184] One hundred fifty isolated islets were transplanted into the subrenal capsule of STZ-induced diabetic mice using a glass micropipette. To prevent immediate loss of islet grafts, a cocktail of reported anti- and pro-inflammatory monoclonal antibodies (mAbs) containing anti-mouse IFN-γ monoclonal antibody (mAb) R4-6A2 (rat IgGκ; e-Bioscience), anti-mouse TNF-α mAb MP6-XT3 (rat IgG1κ; e-Bioscience), and anti-mouse IL-1β mAb B122 (American hamster IgG; e-Bioscience) was intraperitoneally administered three times on days 0, 2, and 4 after transplantation.

[0185] (immunohistochemistry) Two months after islet transplantation, GFP expression (indicating transplanted islets) was observed. The presence of transplanted islets was confirmed by HE staining and DAPI GFP staining of kidney sections.

[0186] (blood glucose monitoring) Blood glucose levels in non-fasting mice were monitored by collecting blood samples at the time of mAb administration and every other week for up to 2 months after islet transplantation. Blood glucose levels were measured using a Medisafe Mini GP-102 (purchased from TERUMO). A glucose tolerance test (GTT) was also performed 2 months after islet transplantation.

[0187] The data are shown in Figure 5A. Figure 5A shows the transplantation of iPS-derived islets into STZ-induced diabetic mice. Panels a and b show the isolation of islets. The iPS-derived pancreas was perfused with collagenase from the common bile duct (arrow in a), and after density gradient centrifugation, EGFP-expressing iPS-derived islets were enriched (b). Panel c shows the kidney capsule 2 months after islet transplantation. The EGFP-expressing spots (arrows) represent transplanted islets. Panel d shows HE staining of kidney sections (left panel) and GFP staining with DAPI (right panel). Panel e shows the transplantation of 150 iPS-derived islets into STZ-induced diabetic mice. The arrows indicate the time points at which an antibody cocktail (anti-IFN-γ, anti-TNF-α, and anti-IL-1β) was administered. Intraperitoneal blood glucose levels were measured every week for up to 2 months after transplantation. STZ-induced diabetic mice transplanted with iPS islets are represented by black triangles (n=6), and STZ-induced diabetic mice not transplanted with iPS islets are represented by black squares (f) shows the glucose tolerance test (GTT) 2 months after islet transplantation.

[0188] The results in Figure 5A show that transplantation of iPS-derived islets improved the symptoms of diabetes, demonstrating the therapeutic efficacy of organ regeneration technology using iPS cells.

[0189] (Example 2: Example in the case of kidney) In accordance with Example 1, kidney organ regeneration was carried out.

[0190] In this example, mouse iPS cells prepared as described above were transplanted as pluripotent cells into knockout mice characterized by kidney defects, and it was examined whether kidney development would occur.

[0191] We used Sall1 knockout mice (kindly provided by Dr. Ryuichi Nishinakamura, Center for Developmental Biology, Kumamoto University) to demonstrate kidney defects. The Sall1 gene is a mouse homolog of the Drosophila anterior-posterior region-specific homeotic gene spalt (sal). Based on pronephric duct induction experiments in Xenopus, the Sall1 gene encodes a 3969-bp protein of 1323 amino acid residues, suggesting its importance in kidney development (Nishinakamura, R. et al., Development, Vol. 128, pp. 3105-3115, 2001, Asajima Laboratory, University of Tokyo). The Sall1 gene has been reported to be expressed in the central nervous system, otic vesicle, heart, limb buds, and anus in addition to the kidney (Nishinakamura, R. et al., Development, Vol. 128, pp. 3105-3115, 2001).

[0192] This Sall1 gene knockout mouse (backcrossed to the C57BL / 6 strain and analyzed) lacks all 10 zinc finger domains present in the molecule due to the deletion of exon 2 and beyond of the Sall1 gene.As a result of this deletion, the ureteric bud does not invaginate into the metanephric mesenchyme, which is thought to result in abnormalities in the early stages of kidney formation (normal individual, Sall1 knockout mouse).

[0193] The genotype of the Sall1 knockout mice used in the experiment was determined in the same manner as for the genotype of the host mice shown in Figure 5. Mouse bone marrow cells were collected, and GFP-negative hematopoietic stem / progenitor cells (c-Kit+, Sca-1+, Lineage marker-: KSL cells) were sorted using a flow cytometer and placed individually into a 96-well plate. These were cultured under cytokine-containing conditions for 12 days to form colonies, from which genomic DNA was extracted and used for genotyping. Furthermore, to confirm the establishment of blastocyst complementation (whether this occurred due to organ vacancies (=knockout (KO))), single-cell genotyping was performed to confirm the KO status by genotyping.

[0194] The primers used for genotyping are as follows: Forward primer for identifying the origin (i.e., host) of injected embryos: For mutant detection: AAG GGA CTG GCT GCT ATT GG (SEQ ID NO: 12) For wild type detection: GTA CAC GTT TCT CCT CAG GAC (SEQ ID NO: 13) Reverse primer for identification of injected embryo origin (i.e., host): For mutant detection: ATA TCA CGG GAT GCC AAC GC (SEQ ID NO: 14) For wild type detection: TCT CCA GTG TGA GTT CTC TCG (SEQ ID NO: 15).

[0195] When produced using this method, the offspring are heterozygous, so the ratio of wild type to heterozygous to KO is expected to be 1:2:1, following Mendelian inheritance. Therefore, to identify the KO individuals, we performed genotyping using bone marrow cells as described above and determined their genotype (Figure 6). It can be seen that mouse #3 was a Sall1 homozygous KO mouse.

[0196] By carrying out such genotyping, it can be confirmed that genotyping is possible in chimeric individuals.

[0197] When kidney formation was examined in mice offspring determined to be homozygous (Sall1(- / -)) or heterozygous (Sall1(+ / -)) by the above genotyping, it was found that kidneys were formed in heterozygotes (Sall1(+ / -)), while kidneys were not formed at all in homozygotes (Sall1(- / -)).

[0198] Male and female heterozygous Sall1 knockout mice (Sall1(+ / -)) were mated, and blastocyst-stage fertilized eggs were collected by uterine reflux. The genotypes of the blastocyst-stage fertilized eggs obtained in this manner are expected to be homozygous (Sall1(- / -)): heterozygous (Sall1(+ / -)): wild-type (Sall1(+ / +)) = 1:2:1.

[0199] The above-mentioned GFP-marked iPS cells were injected into the collected blastocyst-stage fertilized eggs by microinjection at a rate of 15 cells per blastocyst, and the eggs were then transferred back into the uterus of a foster mother (ICR mouse, purchased from Japan SLC Co., Ltd.).

[0200] In the neonatal chimeric individuals confirmed to be homozygous (Sall1(- / -)) by the above genotyping, kidneys were confirmed to be present in the retroperitoneal region. When these formed kidneys were observed under a fluorescent stereomicroscope, GFP-positive findings were confirmed (Figure 6). This indicates that in homozygous (Sall1(- / -)) individuals, the kidneys are derived solely from the mouse iPS cells transplanted into the lumen of the blastocyst-stage fertilized eggs. On the other hand, in heterozygous (Sall1(+ / -)) individuals, the kidneys are composed of a chimera of cells derived from heterozygous (Sall1(+ / -)) individuals and cells derived from the transplanted iPS cells, which could be confirmed by obtaining positive cell images for both GFP fluorescence and fluorescence derived from immunohistochemistry using an anti-GFP antibody.

[0201] Histological analysis of the kidneys obtained by transplanting iPS cells into homozygous (Sall1(- / -)) blastocyst-stage fertilized eggs revealed mature functional glomeruli containing red blood cells within the loop cavity, as well as mature tubular structures. Immunohistochemical analysis using an anti-GFP antibody confirmed that most of these mature cells were GFP-positive.

[0202] From the above, it can be confirmed that in the chimeric Sall1 knockout mice (Sall1(- / -)) produced by the above-mentioned method, the kidneys formed in the offspring were formed from iPS cells transplanted into the lumen of the blastocyst-stage fertilized eggs of Sall1 knockout mice (Sall1(- / -)).

[0203] Example 3: Hair development in hair-deficient mouse strains Regarding hair, we used nude mouse-derived blastocysts and transplanted the mouse iPS cells produced above as pluripotent stem cells to examine whether hair development occurred.

[0204] (Mouse used) The mice used were nude mice obtained from Japan SLC Co., Ltd. The nude mice used were robust and highly reproductively efficient nude mice created by introducing the nu gene of BALB / c nude mice into the inbred DDD / 1 strain of mice.

[0205] Mouse iPS cells were injected into the blastocysts using a micromanipulator under a microscope. The mouse iPS cells used were those transfected with GFP. Equivalently marked mouse iPS cells may also be used. After injection, the embryos were transplanted into the uterus of a foster mother, and offspring were obtained.

[0206] Nude mice are a spontaneously developing model, and although they lack thymus and hair, this does not impair their survival or reproduction. Therefore, mating between nude mice is possible. Therefore, all offspring will be nude mice, and there is no need to determine the genotype. Therefore, confirmation by PCR detection as in the above example is not necessary.

[0207] The growth of hair was confirmed with the naked eye. This is an example of hair growth in nude mice using the method of the present invention. From these results, it was confirmed that the hair that grew was GFP-positive, and that hair can be regenerated using mouse iPS cells.

[0208] (summary) From the above, it was demonstrated that hair can be regenerated even from mouse iPS cells using the method of the present invention.

[0209] Example 4: Thymus development in athymic mouse strains Regarding the thymus, we used nude mouse-derived blastocysts and transplanted the mouse iPS cells produced above as pluripotent cells to examine whether thymus development occurred.

[0210] (Mouse used) The mice used were nude mice obtained from Japan SLC Co., Ltd. The nude mice used were robust and highly reproductively efficient nude mice created by introducing the nu gene of BALB / c nude mice into inbred DDD / 1 strain mice.

[0211] (Mouse maintenance procedures and checks) Mouse iPS cells were injected into the blastocysts under a microscope using a micromanipulator. These mouse iPS cells were transfected with GFP. Equivalently marked mouse iPS cells may also be used. After injection, the embryos were transplanted into the uterus of a foster mother to obtain offspring. In this example, nude mice were used as described in Example 3, so PCR confirmation was not necessary.

[0212] To demonstrate the development of the thymus, we stained CD4- and CD8-positive T cells. This is because the presence of the thymus induces the differentiation of mature T cells, whereas the absence of thymus regeneration leads to the absence of mature T cells. However, when GFP-marked normal iPS cells were transferred into nude mouse blastocysts (BC, blastocyst complementation), both GFP-negative T cells (derived from hematopoietic stem cells of the host nude mouse) and GFP-positive T cells (derived from iPS cells) were induced to differentiate, confirming the functional construction of the thymus by mouse iPS cells.

[0213] Furthermore, to demonstrate the development of the thymus in nude mice, wild-type mice, and chimeric mice of the present invention, we took normal and fluorescent photographs of the thymus of a wild-type mouse, normal and fluorescent photographs of the thymus of a nude mouse, normal and fluorescent photographs of the thymus of a chimeric mouse produced by blastocyst complementation as described above, and fluorescent photographs of the thymus extracted from this chimeric mouse. Confirmation that the thymus exhibited fluorescence demonstrated that it was a tissue derived from mouse iPS cells.

[0214] (summary) From the above, it was demonstrated that the thymus can be regenerated even from mouse iPS cells using the method of the present invention.

[0215] Example 5 In this example, we investigated interspecies blastocyst complementation using Pdx1 knockout mice, which are characterized by pancreatic deficiency, as the host animal and rat iPS cells (EGFP+) prepared according to the above preparation example as the donor cells.

[0216] A. Animals Used As in Example 1, heterozygous Pdx1 gene knockout mice (Pdx1(+ / -)) and homozygous Pdx1(- / -):founder) mice whose pancreases were supplemented with mouse iPS cells were used as knockout mice characterized by pancreatic deficiency.

[0217] B. Preparation of Rat iPS Cells 1) Construction of vectors for generating rat iPS cells The lentiviral vector CS-CDF-CG-PRE contained the TRE from pTRE-Tight (Clontech), the ubiquitin C promoter, the tTA from pTet-on advanced (Clontech), and the IRES2EGFP from pIRES2EGFP (Clontech) in this order from the 5' end. Mouse Oct4, Klf4, and Sox2 were linked with virus-derived F2A and T2A, respectively, and inserted between the TRE and ubiquitin C promoter of the lentiviral vector (LV-TRE-mOKS-Ubc-tTA-I2G).

[0218] 2) Establishment of rat iPS cells: Wistar rat fetal fibroblasts (E14.5) within passage 5 were seeded onto 0.1% gelatin-coated dishes and cultured in DMEM, 15% FCS, 1% penicillin / streptomycin / L-glutamine (SIGMA). The day after seeding, lentivirus prepared using the LV-TRE-mOKS-Ubc-tTA-I2G vector was added to the culture medium for viral infection. After 24 hours, the medium was replaced, and the cells were re-seeded onto mitomycin C-treated MEFs and cultured in DMEM, 15% FCS, 1% penicillin / streptomycin / L-glutamine supplemented with 1 μg / ml doxycycline and 1000 U / ml rat LIF (Millipore). The next day, the medium was changed to serum-free N2B27 medium (GIBCO) supplemented with 1 μg / ml doxycycline and 1000 U / ml rat LIF (Millipore), which was changed every other day. From day 7, inhibitors (2i: 3 mM CHIR99021 (Axon), 1 mM PD0325901 (Stemgent), 3i: 2i + 2 mM SU5402 (CalbioChem)) were added. Colonies that appeared after day 10 were picked and replated onto MEF feeders. The riPS cells established in this way were passaged every 3–4 days using trypsin-EDTA and then transferred into non-human mammalian blastocysts.

[0219] C. Cross-species blastocyst complementation Male Pdx1(- / -) mice were mated with female Pdx1(+ / -) mice, and fertilized eggs were collected by uterine reflux. The collected fertilized eggs were developed in vitro to the blastocyst stage, and the resulting blastocysts were microinjected with the EGFP-marked rat iPS cells described above at 10 cells per blastocyst. These were then transplanted into the uteruses of pseudopregnant foster mothers (ICR mice, purchased from Japan SLC, Inc.). Laparotomy was performed at full term, and the resulting newborns were analyzed.

[0220] Observation of EGFP fluorescence under a fluorescent stereomicroscope revealed that newborn individual numbers #1, #2, and #3 were chimeric, based on EGFP expression on the body surface. Laparotomy revealed that #1 and #2 had pancreases that uniformly expressed EGFP. Meanwhile, the pancreas of #3 partially expressed EGFP, but in a mosaic pattern. Furthermore, #4, a littermate of #1-3, did not exhibit EGFP fluorescence on the body surface, and laparotomy revealed a lack of pancreas, indicating that it was a non-chimeric Pdx1(- / -) mouse (Figure 10).

[0221] In addition, spleens were removed from these newborns, and blood cells extracted from them were stained with mouse or rat CD45 monoclonal antibodies and analyzed by flow cytometry. As a result, rat CD45-positive cells were observed in addition to mouse CD45-positive cells in individual numbers 1 to 3, confirming that these individuals were mouse-rat xenogeneic chimeras, consisting of a mixture of host mouse and rat iPS cell-derived cells. Furthermore, almost all cells in the rat CD45-positive cell fraction exhibited EGFP fluorescence, indicating that the rat CD45-positive cells were derived from EGFP-marked rat iPS cells (Figure 10).

[0222] Furthermore, as an experiment to confirm the establishment of blastocyst complementation (whether this occurred due to the availability of organs (=knockout (KO))), in order to confirm by genotype analysis from single cells that the genotypes of the host mice with individual numbers #1 to #3 were KO, mouse CD45-positive cells were recovered from the spleen samples analyzed with the flow cytometer described above, and genomic DNA was extracted and used for genotype determination.

[0223] The primers used for genotyping were as follows: Forward primer for identification of cells derived from injected embryos: Common to mutant and wild type: ATT GAG ATG AGA ACC GGC ATG (SEQ ID NO: 16) Reverse primer for identification of cells derived from injected embryos: For mutant detection: TTC AAC ATC ACT GCC AGC TCC (SEQ ID NO: 17) For wild type detection: TGT GAG CGA GTA ACA ACC (SEQ ID NO: 18).

[0224] As a result, only mutant bands were observed in #1 and #2, while both mutant and wild-type bands were detected in #3. This indicated that the genotype of the host mice was Pdx1(- / -) in #1 and #2, and Pdx1(+ / -) in #3 (Figure 10A). Based on these results, we successfully constructed a rat pancreas within #1 and #2, Pdx1(- / -) mice, which are normally unable to form a pancreas, by applying the xenogeneic blastocyst complementation technique using rat iPS cells as donors.

[0225] Example 6: Example using animals other than mice This example demonstrates that organs can be produced even when animals other than mice are used. The establishment of pluripotent stem cells capable of forming chimeras in species other than mice can be similarly carried out in accordance with Example 1 by producing iPS cells based on the above preparation example and then producing chimeras.

[0226] Here, iPS cells can be produced in accordance with Example 1 from rats, pigs, cows, and humans instead of mice.

[0227] For example, in this example, it is assumed that similar experiments can be performed with animal species other than mice that are capable of producing genetically modified animals (rats (transgenic), pigs (transgenic, knockout), cattle (transgenic, knockout)) by taking Example 1 into consideration.

[0228] This allows the production of lethal genetically modified founder rats, pigs, cows, etc.

[0229] In this way, similar experiments can be carried out in accordance with Example 1 even when rats, pigs, or cows are used.

[0230] While the present invention has been illustrated by way of preferred embodiments thereof, it is understood that the scope of the present invention should be construed solely in terms of the claims that follow. It is understood that the patents, patent applications, and literature cited herein are incorporated by reference in their entirety as if the contents themselves were specifically set forth herein. [Sequence List Free Text]

[0231] SEQ ID NO: 1: Forward primer for Oct3 / 4, Fw(mOct3 / 4-S1120): CCC TGG GGA TGC TGT GAG CCA AGG SEQ ID NO: 2: Reverse primer for Oct3 / 4, Rv (pMX / L3205): CCC TTT TTC TGG AGA CTA AAT AAA SEQ ID NO: 3: Forward primer for Klf4, Fw(Klf4-S1236): GCG AAC TCA CAC AGG CGA GAA ACC SEQ ID NO: 4: Reverse primer for Klf4, Sox2, and c-Myc, Rv (pMXs-AS3200): TTA TCG TCG ACC ACT GTG CTG CTG SEQ ID NO: 5: Forward primer for Sox2, Fw(Sox2-S768): GGT TAC CTC TTC CTC CCA CTC CAG SEQ ID NO: 6: Forward primer for c-Myc, FW (c-Myc-S1093): CAG AGG AGG AAC GAG CTG AAG CGC SEQ ID NO: 7 Forward (Fw) primer for identification of cells derived from injected embryos: ATT GAG ATG AGA ACC GGC ATG SEQ ID NO: 8 Reverse 1 (Rv1) primer for identification of cells derived from injected embryos: TTC AAC ATC ACT GCC AGC TCC SEQ ID NO: 9 Reverse (Rv2) primer for identification of cells derived from injected embryos: TGT GAG CGA GTA ACA ACC SEQ ID NO: 10 Transgene detection forward (Fw) primer: TGA CTT TCT GTG CTC AGA GG SEQ ID NO: 11 Reverse (Rv) primer for detecting transgene: CAA TGA TGG CTC CAG GGT AA SEQ ID NO: 12 Forward primer for detecting cells (mutant) derived from injected embryos: AAG GGA CTG GCT GCT ATT GG SEQ ID NO: 13 Forward primer for detecting cells derived from injected embryos (wild type): GTA CAC GTT TCT CCT CAG GAC SEQ ID NO: 14 Injected embryo derived cells (mutant) reverse primer: ATA TCA CGG GAT GCC AAC GC SEQ ID NO: 15 Reverse primer for detecting cells derived from injected embryos (wild type): TCT CCA GTG TGA GTT CTC TCG SEQ ID NO: 16 Forward primer for detecting cells derived from injected embryos (mutant and wild type): ATT GAG ATG AGA ACC GGC ATG SEQ ID NO: 17 Reverse primer for detecting cells (mutant) derived from injected embryos: TTC AAC ATC ACT GCC AGC TCC SEQ ID NO: 18 Reverse primer for detecting cells derived from injected embryos (wild type): TGT GAG CGA GTA ACA ACC

Claims

1. 1. A method for producing a target organ or body part derived from a heterologous mammal, which is an individual different from a non-human mammal, in a living body of a non-human mammal having an abnormality that prevents the development of the target organ or body part at a developmental stage, the method comprising: a) preparing induced pluripotent stem cells (iPS cells) derived from the allogeneic mammal; b) implanting the cells into a blastocyst stage fertilized egg of the non-human mammal; c) allowing the fertilized egg to develop in the womb of a non-human foster mammal to obtain a litter; and d) obtaining the target organ or body part from the offspring. Including, the iPS cells and the non-human mammal are of different species; A method for producing a target organ or body part.

2. 2. The method of claim 1, wherein the iPS cells are derived from a human, a rat, or a mouse.

3. The method of claim 1, wherein the iPS cells are derived from a rat or a mouse.

4. 10. The method of claim 1, wherein the organ or body part to be produced is selected from the group consisting of pancreas, kidney, thymus, and hair.

5. The method of claim 1 , wherein the non-human mammal is a mouse.

6. The method of claim 5, wherein the mouse is a Sall1 knockout mouse, a Pdx1-Hes1 transgenic mouse, a Pdx-1 knockout mouse, or a nude mouse.

7. The method according to claim 1, wherein the target organ is entirely derived from the allogeneic mammal.

8. The method of claim 1, further comprising the step of obtaining the iPS cells by contacting somatic cells with reprogramming factors.

9. The method of claim 1, wherein the iPS cells are derived from a rat and the non-human mammal is a mouse.

10. A non-human mammal having an abnormality in which a target organ does not develop during the developmental stage, and having the target organ or body part derived from a different individual mammal, a) preparing iPS cells derived from a heterologous mammal that is different from the non-human mammal (wherein the iPS cells and the non-human mammal are of different species); b) transplanting the iPS cells into a blastocyst-stage fertilized egg of the non-human mammal having an abnormality that prevents the development of a target organ during development; and c) allowing the fertilized egg to develop in the womb of a non-human foster mammal to obtain offspring. A mammal produced by a method comprising:

11. Use of a non-human mammal having an abnormality that prevents the development of a target organ during the developmental stage for producing the target organ using iPS cells, wherein the iPS cells and the non-human mammal are of different species.

12. A set for producing a target organ, the set comprising: A) a non-human mammal having an abnormality in which the target organ does not develop during the developmental stage; B) iPS cells derived from a heterologous mammal of an individual different from the non-human mammal, or reprogramming factors and, if necessary, somatic cells derived from a heterologous mammal of an individual different from the non-human mammal; the iPS cells and somatic cells are of a heterologous relationship with the non-human mammal; set.

13. 1. A method for producing a desired organ or body part, comprising: A) providing a non-human mammal comprising a deficiency-causing gene encoding a deficiency cause of an organ or body part that would be non-viable or difficult to survive if it functioned, and the organ or body part is complemented by blastocyst complementation, wherein the deficiency-causing gene encodes a deficiency cause of the organ or body part of interest; B) obtaining eggs from said animal and developing them into blastocysts; C) introducing into the blastocyst a target iPS cell having a desired genome capable of complementing the defect caused by the defective gene, thereby producing a chimeric blastocyst (wherein the iPS cell and the non-human mammal are of different species); and D) Producing an individual from the chimeric blastocyst and obtaining the desired organ or body part from the individual. A method that encompasses

14. The method according to claim 13, further comprising the step of obtaining the iPS cells by contacting somatic cells with reprogramming factors.

15. The method according to claim 13, wherein step D) comprises developing the chimeric blastocyst in a maternal womb of a non-human foster mammal to obtain a litter, and obtaining the target organ from the litter.

16. The method according to claim 13, wherein the target iPS cells are derived from a rat or a mouse.

17. 14. The method of claim 13, wherein the organ or body part of interest is selected from the group consisting of pancreas, kidney, thymus, and hair.

18. The method of claim 13, wherein the animal is a mouse.

19. The method of claim 18, wherein the mouse is a Sall1 knockout mouse, a Pdx-1 knockout mouse, a Pdx1-Hes1 transgenic mouse, or a nude mouse.

20. The method according to claim 13, wherein the organ or body part of interest is entirely derived from the iPS cells of interest.

21. The method of claim 13, wherein the iPS cells are derived from a rat and the non-human mammal is a mouse.

22. A set for producing a target organ or body part, the set comprising: A) A non-human mammal comprising a gene encoding a cause of a defect in an organ or body part that would be non-viable or difficult to survive if it functioned, and the organ or body part is to be complemented by complementation; B) iPS cells derived from a heterologous mammal of an individual different from the non-human mammal, or reprogramming factors and, if necessary, somatic cells derived from a heterologous mammal of an individual different from the non-human mammal. The combination of the iPS cells and somatic cells are heterologous to the non-human mammal; set.

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