WO2010143747A1

WO2010143747A1 - Method for production of artificial intestinal tract

Info

Publication number: WO2010143747A1
Application number: PCT/JP2010/060254
Authority: WO
Inventors: 中島祥介; 山田高嗣; 植田剛
Original assignee: 公立大学法人奈良県立医科大学
Priority date: 2009-06-10
Filing date: 2010-06-10
Publication date: 2010-12-16

Abstract

A method for producing an artificial intestinal tract from an artificial pluripotent stem (iPS) cell, which comprises forming an embryoid body from a purified iPS cell using a three-dimensional culture system, culturing the embryoid body, and culturing the embryoid body using a two-dimensional attached culture system, thereby inducing the differentiation into an intestinal tract; an artificial intestinal tract produced by the method; and use of the artificial intestinal tract in regenerative medicine.

Description

How to make an artificial intestine

The present invention relates to a method for producing an artificial intestinal tract from induced pluripotent stem (iPS) cells.
The present invention also relates to an artificial intestinal tract produced by the above-described method and use of the artificial intestinal tract for regenerative medicine.

Today, regenerative medicine is in the spotlight. This is because iPS cells were discovered by Yamanaka et al. (Patent Document 1, Non-Patent Document 1). iPS cells have characteristics similar to embryonic stem (ES) cells derived from somatic cells by the action of specific transcription factors, and are capable of differentiating into various cells and tissues ( Non-patent documents 1 to 3). Since ES cells are derived from eggs or oocytes, iPS cells can be derived from individual somatic cells, so that when differentiated cells or tissues derived from iPS cells are transplanted into an individual, There is an advantage that there is almost no risk of rejection. Therefore, research for using iPS cells for regenerative medicine is being conducted.
ES cells were first induced from mouse fertilized eggs (Non-patent Document 4), and human ES cells were also established thereafter (Non-patent Document 5). It has been reported that ES cells, particularly mouse ES cells, induce differentiation into cells such as vascular endothelial cells, smooth muscle cells, cardiomyocytes, nerve cells, insulin producing cells, dopamine producing cells (Non-patent Document 6). ~ 9). In contrast, iPS cell utilization research has just begun, and there are few reports on induction from iPS cells to differentiated cells.
Regarding the artificial intestinal tract related to the present invention, construction of an intestinal tract-like cell mass from mouse ES cells (Patent Document 2), induction of intestinal differentiation from mouse ES cells (Non-Patent Documents 10 to 11), and the like have been reported. In this report, an intestinal tract that is peristaltically moved by subjecting an embryoid body formed from ES cells in a hanging drop culture system to adherent culture has been prepared. It has been pointed out that culture conditions are an important factor.
However, since epigenetic studies have reported that iPS cells are not identical to ES cells (Non-patent Document 12), and because iPS cells themselves are not sufficiently characterized, ES cell information remains as is in iPS. There is no guarantee that it can be applied to cells. Under such circumstances, if induction of differentiation of the intestinal tract peristalized from iPS cells is realized, the expectation of application to regenerative medicine will be further increased.

Japanese Patent No. 4183742 Japanese Unexamined Patent Publication No. 2006-239169

When an artificial pluripotent stem (iPS) cell is transplanted into a nude mouse, it forms a teratoma, and thus iPS cells are considered to have pluripotency and theoretically differentiate into various cells. Is expected. However, unlike the pluripotency of ES cells established from fertilized eggs, iPS cell differentiation pluripotency produced by gene transfer of a specific type of reprogramming factor into somatic cells such as mammalian skin cells Regarding sex, there is no reliable information on whether to differentiate into all cells, tissues, organs, etc. that make up the body.
The present inventors previously reported a method for inducing differentiation of an artificial intestinal tract from embryonic stem (ES) cells. This time, the present inventors have studied the production of an artificial intestinal tract from iPS cells, which is considered to have a wider application range. went.
Accordingly, an object of the present invention is to provide a method for producing an artificial intestinal tract from iPS cells.
In summary, the present invention includes the following features.
In a first aspect, the present invention relates to a method for producing an artificial intestinal tract from artificial pluripotent stem (iPS) cells, which uses a three-dimensional three-dimensional culture system to form and culture embryoid bodies from purified iPS cells. And then culturing the embryoid body using a two-dimensional adhesion culture system, thereby inducing differentiation of the intestinal tract.
In the embodiment, the three-dimensional three-dimensional culture system is a suspension culture system.
In another embodiment, the iPS cells are derived from mammalian somatic cells.
In another embodiment, the embryoid body is formed and cultured for 5 to 7 days, preferably 6 days before the two-dimensional adhesion culture.
In another embodiment, in the suspension culture system, the number of cells per drop (about 15-30 μl) is about 400-3,000, for example about 500-2,000, preferably one drop (about 15 μl). The number of cells per cell is 500 to 1,000.
In another embodiment, the intestinal tract has a neural network.
The intestinal tract produced by the method of the present invention is composed of intestinal components derived from the three germ layers and is characterized by peristaltic movement.
Therefore, in the second aspect, the present invention also provides an artificial intestinal tract produced by any of the methods described above.
In a third aspect, the present invention further provides the use of the artificial intestinal tract described above for the manufacture of a therapeutic agent for regenerative medicine resulting from a patient's bowel disease.
The present invention provides an advantage that an artificial intestinal tract can be prepared using iPS cells derived from a patient's somatic cells, thereby avoiding the risk of rejection associated with transplantation. This also has the advantage that the range of application is greatly expanded compared to the production of an artificial intestine from embryonic stem (ES) cells.

FIG. 1 shows a tubular formation artificial tube derived from iPS cells.
FIG. 2 shows a dome-like artificial intestinal tract induced to differentiate from iPS cells. There are many types of artificial intestinal tracts induced to differentiate from ES cells, but artificial intestinal tracts induced to differentiate from iPS cells tend to be more in the tubular formation of FIG.
FIG. 3 shows the delivery function (FIGS. 3A to 3C) of the intestinal contents (black) accompanying peristaltic movement (repetitive contraction and relaxation) in the artificial intestine of the present invention, and enlarged images (FIGS. 3D to 3F).
FIG. 4 shows the presence of nerve cell bodies and nerve fibers of nerve cells differentiated from iPS cells in the artificial intestinal tract of the present invention (FIG. 4A) and the ganglion that is an aggregate of large nerve cells (FIG. 4). 4B). In the figure, the scale bar represents 30 μm.

1. Definitions The definitions of terms used in this specification are described below. However, these definitions should not be interpreted in a limited way, but in the broadest sense used in the industry.
The artificial intestinal tract refers to a peristaltic intestinal tract that is induced to differentiate from an induced pluripotent stem (iPS) cell. Components constituting the intestinal tract include those derived from the three germ layers (ie, ectoderm, endoderm and mesoderm), such as nerve cells, mucosal epithelial cells, microvilli, connective tissue, smooth muscle layer and the like.
An induced pluripotent stem cell or iPS cell refers to an ES cell-like differentiated pluripotent stem cell initialized from a somatic cell by a reprogramming factor. The cells express ES cell specific markers such as Oct3 / 4, Nanog (or OCT3 / 4, NANOG). iPS cells also have the ability to differentiate into the various cells that make up the animal's body and to continue to proliferate semi-permanently while retaining their karyotype.
Reprogramming refers to the process and means by which differentiated cells are induced and converted to undifferentiated cells, particularly differentiated pluripotent cells.
A three-dimensional three-dimensional culture system is a three-dimensional culture system that is not a two-dimensional (planar) culture. For example, suspension culture (Keller, J., Physiol. (Lond) 168: 131-139 (1998), JP, 2008-178367 publication), suspension culture (same as above), three-dimensional culture system under microgravity environment (T. Uemura et al., Space Utility Res 25: 170-173 (2009)) and the like. Suspension culture is also referred to as hanging drop culture, and is a culture method that utilizes gravity and surface tension, and is a method of culturing cells in a culture solution that hangs down in the form of water droplets. The suspension culture is a culture method in which cells are grown in a suspended state in a liquid medium without being attached to a culture vessel by coating with a cell non-adhesive polymer. In suspension culture, a previously formed embryoid body or aggregate (or aggregate) can be suspended and cultured. It is known that three-dimensional culture in a microgravity environment can be performed using a RWV (rotating wall vessel) bioreactor, and three-dimensional tissue construction is possible.
A feeder cell is an auxiliary cell used to prepare a culture condition in which a cell to be proliferated is kept undifferentiated, and this cell itself is an antibiotic such as mitomycin C or radiation so as not to proliferate. Processed in advance by irradiation or the like.
An embryoid body is a cell cluster formed by aggregation of iPS cells.
Mammals include, but are not limited to, primates including humans, rodents including mice, and ungulates including cows. A preferred mammal is a human.
Somatic cells are all somatic cells except germ cells (oocytes, spermatogonia), germ stem cells (embryonic stem cells, sperm stem cells) or totipotent cells, mature or fetal cells (fibroblasts) Hair cells, muscle cells, hepatocytes, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells, pancreatic exocrine cells, brain cells, lung cells, kidney cells, skin cells, lymphocytes, epithelial cells, endothelial cells, etc.), It includes cells such as stem cells (hematopoietic stem cells, mesenchymal stem cells, neural stem cells, dental pulp stem cells, etc.) and precursor cells. Somatic cells also include cells such as primary cultured cells, passaged cells, and established cell lines.
The tridermal system consists of endoderm, mesoderm, and ectoderm, and can differentiate into specific cells, tissues, and organs, respectively.
The intestinal tract component refers to cells derived from the three germ layers that constitute the intestinal tract.
Peristaltic movement refers to cooperative movement unique to the intestinal tract.
2. Production of iPS cells The iPS cells that can be used in the present invention can be artificially induced by reprogramming of somatic cells of any animal, preferably mammals, including humans and mice (for example, Takahashi, K. and Yamanaka, S., Cell 126: 663-676 (2006), Takahashi, K. et al., Cell 131: 861-872 (2007), Yu, J. et al., Science 318: 1917-1920 (2007)). . Reprogramming is not particularly limited, and can be performed by any known method. Such methods include methods that use a combination of specific reprogramming factors, methods that combine reprogramming factors with microRNA (miRNA), and the like.
Examples of combinations of reprogramming factors are not limited to the following, but combinations of Oct3 / 4, Sox2, Klf4 and c-Myc, or OCT3 / 4, SOX2, KLF4 and c-MYC, or homologs thereof. , Oct3 / 4, Sox2 and Klf4, or OCT3 / 4, SOX2 and KLF4, or a homologue thereof, Oct3 / 4, Sox2, Nanog and Lin28, or OCT3 / 4, SOX2, NANOG and LIN28, or a combination thereof Homologs, etc. (Takahashi, K. and Yamanaka, S., Cell 126: 663-676 (2006), Takahashi, K. et al., Cell 131: 861-872 (2007), Yu, J. et al. et al., Science 318: 1917-1920 (2007)). These reprogramming factors are either nucleic acids (ie, genes, DNA or RNA) or proteins.
The amino acid sequence and base sequence of the reprogramming factor can be obtained, for example, by accessing the web site of NCBI, USA. For example, for Oct3 / 4, Sox2, Klf4, c-Myc, Nanog, and Lin28, the mouse sequences (amino acid sequence and base sequence) are registered as NM_013633, NM_011433, NM_010638, NM_010844, NM_028016, NM_145833, respectively. In addition, for OCT3 / 4, SOX2, KLF4, c-MYC, NANOG and LIN28, human sequences (amino acid sequence and base sequence) are registered as NM_203289 or NM_002701, NM_003106, NM_004235, NM_002467, NM_024865, NM_024647, respectively. ing.
Examples of combinations of reprogramming factors and miRNAs are not limited to the following, but include Oct3 / 4, Sox2 and Klf4, or OCT3 / 4, SOX2 and KLF4, or homologs thereof, and miR-291-3p, miR -294 or miR-295, or combinations thereof with homologs of those miRNAs (Judson, RL et al., Nature Biotechnology 27: 459-461 (2009)). miR-291-3p, miR-294, or miR-295 are subsets of the mouse miR-290 cluster that increase the efficiency of somatic cell reprogramming by Oct3 / 4, Sox2 and Klf4 upon induction of mouse iPS cells. Has an enhancing effect.
As used herein, the term “homolog” is used when it belongs to the same family as the indicated reprogramming factor or miRNA, but the species from which it is derived is different.
Reprogramming is performed by introducing a reprogramming factor or miRNA into the somatic cell. When the reprogramming factor is a nucleic acid, it can be introduced into a somatic cell via a vector such as a virus or a plasmid. Viral vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors, and the like. In addition, any plasmid for mammalian cells can be used as the plasmid (Okita, K. et al., Science 322: 949-953 (2008)). Among these, when an adenovirus vector, an adeno-associated virus vector or a plasmid is used, the reprogramming factor DNA is not integrated into the cell genome (Stadfeld, M. et al., Science 322: 945-949 (2008)). In addition, when Sendai virus vector is used, it is said that the vector is destroyed after reprogramming.
In addition to the reprogramming factor DNA, the vector may appropriately include a control element such as a promoter, enhancer, terminator, ribosome binding site, polyadenylation site, selection marker, reporter gene, and the like. Selectable markers include, for example, drug resistance genes such as positive markers such as kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, and negative markers such as thymidine kinase gene and diphtheria toxin gene. The reporter gene includes gene sequences such as green fluorescent protein (GFP), GUS (β-glucuronidase), and FLAG.
Introduction of the reprogramming factor into the cell can be performed by a method utilizing viral infection, electroporation, cationic liposome, membrane-permeable peptide vector (WO2007 / 132555), membrane-permeable peptide surface-modified liposome, or the like. . In particular, liposomes and membrane-permeable peptide vectors can be used for intracellular introduction of reprogramming factor proteins.
General techniques for genetic recombination, including vector construction, are described in, for example, Sambrook, J. et al. Et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press (1989), Ausubel, F. et al. M.M. Et al., Short protocols in Molecular Biology: A Comprehensive Methods from Current Protocols in Molecular Biology, John Wiley & Sons (1999).
Induction of iPS cells is described in Japanese Patent No. 4183742, Takahashi, K. et al. and Yamanaka, S .; , Cell 126: 663-676 (2006), Takahashi, K .; et al. , Cell 131: 861-872 (2007) and the like.
One example of iPS cell induction is by culturing somatic cells on feeder cells (mitomycin C-treated STO cells or SNL cells) in an appropriate induction medium in the presence of 37 ° C., 5% CO ₂ , A somatic cell and a reprogramming factor or DNA (preferably a vector) encoding the factor are brought into contact and cultured, and an iPS-like colony formed after about 2 to 3 weeks is identified and selected.
Another example of the iPS cell induction method is to first contact a somatic cell with a reprogramming factor or DNA encoding the factor (preferably a vector) in an appropriate culture medium in the presence of 37 ° C. and 5% CO _2. For about 4-7 days, after which the cells are cultured on the same feeder cells in the presence of 37 ° C., 5% CO ₂ in an appropriate culture medium for about 25 to about 30 days or more. IPS-like colonies generated after the above are identified and selected.
Examples of the culture medium include DMEM, DMEM / F12, or DME medium containing 10 to 15% FCS (there are LIF (leukemia inhibitory factor), penicillin / streptomycin, puromycin, L-glutamine, non-essential). Amino acids, β-mercaptoethanol, etc. can be included as appropriate.), Or ES cell culture medium containing bFGF or SCF, for example, mouse ES cell culture medium (eg, TX-WES medium, Thrombo X) or A culture medium for primate ES cell culture (for example, culture medium for primate (human and monkey) ES cells, reprocell), and the like can be used.
During the above culture, the medium is replaced with a fresh medium once a day from the second day onward. The number of somatic cells used for reprogramming is not limited, but ranges from about 5 × 10 ³ to about 5 × 10 ⁶ cells per 100 cm ^{2 of} culture dish.
By treating the iPS-like colonies on the dish with a solution containing trypsin and collagenase IV (CTK solution), the remaining colonies are spread on the feeder cells and cultured in the ES cell culture medium in the same manner. iPS cells can be passaged. At this time, the cells are cultured until they become 80-90% confluent, and the passage is repeated.
Identification of iPS cells can be performed by detecting the expression of genes such as Oct3 / 4 or OCT3 / 4, Nanog or NANOG, which are ES cell marker genes, by RT-PCR. Furthermore, the formation of teratomas (teratomas) is confirmed by transplanting established cells to the dorsal ventral side of nude mice.
3. Preparation of embryoid bodies from iPS cells When preparing embryoid bodies from iPS cells, it is necessary to purify the iPS cells with high purity. The iPS cells produced and subcultured by the above-mentioned method contain a mixture of feeder cells such as fibroblasts (for example, mouse fetal fibroblasts (MEF)), and a few differentiated cells generated during repeated passages. Therefore, it is important to remove such cells. In order to remove the mixed cells, for example, iPS cells are seeded on gelatin and cultured repeatedly to obtain purified iPS cells. At this time, in order to maintain an undifferentiated state while removing MEF, LIF is contained in the iPS cell culture medium.
Specifically, on fibroblasts (feeders), iPS cells (about 5 × 10 ³ to about 5 × 10 ⁶ cells per 100 cm ^{2 of} culture dish) in the above culture medium in the presence of 37 ° C. and 5% CO ₂ In order to remove fibroblasts from the grown iPS cell colonies, the iPS cells were seeded on gelatin, cultured in iPS cell culture medium for 2 days, and again, iPS cells were cultured on gelatin. Incubate for 1 day. High-purity iPS cells can be obtained in about 5 days from the start of iPS cell culture.
The purity of iPS cells that can be used in the present invention is 95% or more, preferably 97% or more, more preferably 99% or more. As used herein, “purified iPS cells” refers to cells containing 95% or more of iPS cells.
Embryoid bodies are prepared using the purified iPS cells obtained as described above. When there is much MEF contamination and the purity of iPS cells is low, the differentiation induction efficiency of the artificial intestinal tract becomes extremely low. Therefore, purification of iPS cells, that is, removal of feeder cells such as MEF is very important for the production of artificial intestinal tract. It became clear that. At this time, a three-dimensional three-dimensional culture system or a two-dimensional planar culture system can be used as the culture system, but a three-dimensional three-dimensional culture system or a combination of three-dimensional three-dimensional culture and two-dimensional planar culture is preferable. Examples of the three-dimensional three-dimensional culture system include a suspension culture system, a suspension culture system, and a three-dimensional culture system in a microgravity environment. A preferred culture system is a suspension culture system (Keller et al., J. Physiol. (Lond) 168: 131-139 (1998), Japanese Patent Application Laid-Open No. 2006-239169).
The suspension culture system is a culture system in which a culture solution is hung on the back of a petri dish lid, etc., and iPS cells aggregate under the culture drop under the influence of gravity, and an aggregate called an embryoid body becomes a short day. Formed with. In this culture system, it is desirable to determine the number of iPS cells according to the volume of the culture drop, for example, about 400 to 3,000 cells, for example, about 500 to 2,000 cells per drop (about 15 to 30 μl), preferably Is a suitable cell concentration of about 500 to 1,000 cells per drop (about 15 μl).
A medium for producing an embryoid body in the above culture system is, for example, based on a DMEM, DMEM / F12 or DME medium containing about 10 to 15% FCS, and includes a non-essential amino acid, β-mercapto. Those containing ethanol, sodium pyruvate, penicillin / streptomycin, etc. can be used. A medium similar to this can be used in a suspension culture system or a three-dimensional culture system in a microgravity environment.
By suspension culture as described above, embryoid bodies obtained by culturing iPS cells for 5 to 7 days, preferably 6 days are obtained. It is now clear that the number of days of embryoid body culture is very important for the development of the artificial intestinal tract because the induction of differentiation of the artificial intestinal tract is higher in 6 days than in 5 days. became.
4). Induction of differentiation of artificial intestinal tract The embryoid body obtained by culturing iPS cells as described above for 5 to 7 days, preferably 6 days, is cultured for about 14 days using the same medium as that for embryoid body preparation. By performing two-dimensional adhesion culture, it is possible to induce differentiation of the peristaltic intestinal tract having a luminal structure (see FIGS. 1 and 3).
As a group of cells constituting the induced intestinal tract, mucosal epithelial cells such as Goblet cells having microvilli, connective tissue, smooth muscle cells, serosa, Kahal stromal cells, nerve cells, etc. were detected. A group of cells from the germ layer. In addition to regular automatic contraction, a large waving and undulating peristaltic movement is characteristic of the intestinal tract.
From the morphological point of view, the artificial intestinal tract (ES-Gut) from ES cells has many domestic-like formations (domestic shape), and the number of tubular formations (tubular) is small, whereas the iPS cells of the present invention In the artificial intestinal tract (iGut) from, many of them took a tubular formation (tubular) (FIG. 1) rather than a dome-like formation (dome-shaped) (FIG. 2). Here, the dome-shaped tissue is thought to be a tissue formed by cells proliferating and accumulating vertically, whereas the tubular tissue is thought to be formed by cells proliferating and accumulating horizontally. It is unclear whether this difference is due to differences in cell characteristics between ES cells and iPS cells or due to differences at the stage of the internal microenvironment in the embryoid body.
As a result, the artificial intestine from iPS cells could be obtained under the same culture conditions as when the artificial intestine was derived from embryonic stem (ES) cells. However, with regard to human ES cells, there is an ethical problem of destroying fertilized eggs at the stage of preparing human ES cells, and even if such problems are solved, because of rejection reactions associated with transplantation, However, there are problems such as being limited to use only for fertilized egg providers. On the other hand, in the method of the present invention, since somatic cells of the patient are used as starting materials, it is considered that there are no ethical problems and problems of rejection as described above. Certainly, iPS cells are similar to ES cells in terms of characteristics such as morphology, proliferation mode, differentiation pluripotency, and ability to form teratomas, but differentiation from iPS cells into somatic cells and tissues. Actually, there is not enough knowledge about induction. In particular, it is completely unknown whether iPS cells produced by reprogramming somatic cells have the ability to induce organ differentiation. Under such circumstances, it is unpredictable and epoch-making that an organ called an artificial intestine, which is a complex aggregate of cells unique to the intestine, can be induced to differentiate from iPS cells under the same culture conditions as ES cells.
5). Medical Application The present invention further provides the use of the artificial intestinal tract described above for the manufacture of a therapeutic agent for regenerative medicine resulting from a patient's bowel disease.
IPS cells are established from primary or subcultured somatic cells collected from patients in a strictly controlled clean room free from contamination by viruses, microorganisms, etc., and prepared from the iPS cells by the method of the present invention. The produced artificial intestinal tract is used as a therapeutic agent for regenerative medicine. The therapeutic agent includes at least an artificial intestinal tract, a medium for maintaining it (including serum if necessary), artificial blood, and the like.
Intestinal diseases include refractory or congenital diseases such as inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, and intestinal motility disorders such as Hirschsprung's disease.
Since the artificial intestinal tract of the present invention is derived from the patient's own somatic cells, it has the potential to become a completely new therapeutic strategy that can be used for regenerative medicine and tailor-made medicine as an organ for transplantation without rejection.
According to the method of the present invention described above, an artificial intestinal tract is made outside the body, which is replaced with or transplanted to the impaired intestinal tract of a patient. This is thought to cause the patient's intestine to function normally.
Furthermore, by creating an artificial intestinal tract from patient-derived or disease-specific iPS cells, elucidation of the pathophysiology of intestinal diseases, evaluation of drug effects and toxicity, absorption of nutrients from the intestine, creation of infectious enteritis models In addition, it is considered to be useful for clinical application to drug discovery and cell transplantation therapy.
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by these examples.

[Example 1]
<Preparation of embryoid bodies from iPS cells>
iPS cells The Kyoto University iPS Cell Research Center introduced 4 reprogramming factor (Oct3 / 4, Sox2, Klf4, c-Myc) DNA into mouse skin cells (somatic cells) via pMXs retroviral vectors. The iPS cells prepared in this way were provided by Kyoto University through RIKEN. This mouse iPS cell was used in the following experiments.
Maintenance culture of iPS cells The iPS cells donated from Kyoto University were thawed and dispensed, and then stored frozen at -180 ° C. In order to maintain undifferentiated iPS cells, MEPS (mouse fetal fibroblasts) that release undifferentiated factors were used as feeder cells, and iPS cells were cultured thereon. The culture solution at that time is shown in Table 1 below.

Production of Embryoid Body A three-dimensional three-dimensional culture system was used to induce cell-to-organization from iPS cells to complex organs composed of various cells in an orderly manner. Specifically, embryoid bodies were prepared from iPS cells using a hanging drop culture system in which a culture solution containing iPS cells was hung on the back of a petri dish lid. In this culture system, iPS cells aggregated under the culture drop under the influence of gravity, and aggregates called embryoid bodies were formed in one day.
As described above, iPS cells are maintained and cultured, and frozen iPS cells are thawed and used for experiments, in which mouse fetal fibroblast (MEF) cells are contaminated. It has now been clarified that when intestinal differentiation is induced, differentiation is inhibited by mixing these MEF cells with iPS cells. Therefore, it was necessary to remove MEF cells to make iPS cells as pure as possible. Therefore, using the property that MEF easily adheres to gelatin, MEF was removed by culturing several times on a petri dish coated with iPS + MEF.
Actually, the procedure for producing an embryoid body is as follows.
(1) Thaw frozen MEF and culture. The culture solution is the same as in Table 1 above. This operation is performed the day before thawing iPS cells.
(2) Thaw iPS cells and culture on MEF for 2 days.
(3) iPS cells proliferate while forming colonies and maintaining an undifferentiated state. Next, in order to remove MEF, iPS cells + MEF cells are seeded on gelatin and cultured for 2 days.
(4) Again, the culture solution of (3) is spread on gelatin and cultured for 1 day, thereby further removing MEF and increasing the purity of iPS cells.
(5) Only high-purity iPS cells can be cultured in about 5 days from the start of iPS cell culture, and embryoid bodies (EBs) are produced using the purified iPS cells.
As a condition necessary for inducing differentiation of the intestinal tract, it became clear that the optimal concentration of iPS cells was very important at the stage of EB production. Specifically, a culture solution for EB production (Table 2) EBs were produced in a suspension culture system at a concentration of 500 cells / 15 μl.

[Example 2]
<Induction of intestinal differentiation>
After culturing for 6 days in the state of an embryoid body, after performing two-dimensional growth culture, the intestinal tract that peristally moves has been induced to induce differentiation on about the 14th day. The culture solution at this time is the same as in Table 2. When embryoid bodies cultured for 5 days were used, the differentiation induction efficiency of the intestinal tract decreased. For this reason, it was found that 6-day embryoid body culture is desirable.
An artificial intestinal tract is shown in FIG. The shape of an organ having a tubular structure (tubular formation) is clear. Artificial intestinal tracts induced to differentiate from ES cells have many types of dome-like formation (dome shape), but artificial intestinal tracts induced to differentiate from iPS cells are more likely than domes-like formation (dome shape) (FIG. 2). There was a tendency for more tube formation (tubular). In addition, the produced artificial intestinal tract repeatedly contracted and relaxed, and as shown in FIG. 3, the contents of the intestinal tract (black; confirmed with an electron microscope were epithelial cells as a result of this peristaltic movement. Probably the epithelial cells that have fallen off due to regeneration). In addition, by histological and cytological examinations, the cell group constituting the intestinal tract includes mucosal epithelial cells such as Goblet cells having microvilli, connective tissue, smooth muscle cells, serosa, Kahal stromal cells, nerve cells, etc. It has been shown. For nerve cells, nerve staining using an anti-neurofilament antibody revealed that a network of nerve cell bodies, ganglia, and nerve fibers was constructed in the artificial intestine (FIG. 4).
Thus, due to characteristics such as peristaltic movement and intestinal constitutive cell groups, organs induced to differentiate from iPS cells have morphological characteristics similar to those of a mature mouse intestinal tract having a luminal structure and a neural network. Involved in the induction of nerve cell differentiation, the formation of ganglia and nerve fiber networks in the peristaltic movement and transport function.
Surprisingly, starting from iPS cells under the simple culture conditions as described above, an organ that is a peristaltic artificial intestine that was not a mere somatic cell or tissue was produced.

The present invention provides a method for producing an artificial intestinal tract using iPS cells derived from a somatic cell of a patient. An artificial intestine without rejection produced by this method is used for regenerative medicine and tailor-made medicine. Useful for.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

A method for producing an artificial intestinal tract from an induced pluripotent stem (iPS) cell, wherein an embryoid body is formed and cultured from a purified iPS cell using a three-dimensional three-dimensional culture system, and then a two-dimensional adhesion culture system is used. And culturing the embryoid body, thereby inducing differentiation of the intestinal tract.
The method according to claim 1, wherein the three-dimensional three-dimensional culture system is a suspension culture system.
The method according to claim 1 or 2, wherein the iPS cells are derived from mammalian somatic cells.
The method according to any one of claims 1 to 3, wherein the embryoid body is formed and cultured for 5 to 7 days, preferably 6 days, before the two-dimensional adhesion culture.
5. The suspension culture system according to any one of claims 2 to 4, wherein the number of cells per drop (about 15 to 30 μl) is about 400 to 3,000, preferably about 500 to 2,000. Method.
The method according to any one of claims 1 to 5, wherein the intestinal tract has a neural network.
An artificial intestinal tract produced by the method according to any one of claims 1 to 6.
Use of the artificial intestinal tract according to claim 7 for the manufacture of a therapeutic agent for regenerative medicine resulting from a patient's bowel disease.