JP2021069432A - Graft material and stent - Google Patents

Abstract

To provide a graft material that can keep a renal function for a long period of time even if a kidney that a patient originally has is retained, and a stent used for the material.SOLUTION: A graft material comprises: a kidney primordium; a bladder connected to the kidney primordium; and a stent deformable in a spread out fan-shape, in which the bladder is held by the stent which is inflated in a spread out fan-shape after being inserted into the bladder. The graft material is used in order to be grafted such that a surface contacting a ureter of a host is connected in a spread state.SELECTED DRAWING: None

Description

The present invention relates to implantable materials and stents.

The number of dialysis patients is increasing rapidly due to the aging of the population and the expansion of indications. Although it is possible to save the lives of these patients by dialysis treatment, since dialysis cannot compensate for all renal functions, the cardiovascular disease of the patients tends to increase and the case fatality rate tends to increase. In addition, the time and mental burden on dialysis patients is extremely large, and it is often difficult to reintegrate into society.

In addition, there are about 2 million patients with end-stage renal disease requiring kidney transplantation worldwide, and the number tends to increase further due to the shortage of donor organs. Therefore, end-stage renal disease is a serious medical problem.

Against this background, the inventors have developed a transplant material capable of sustaining renal function for a long period of time (see Patent Document 1).

Japanese Patent No. 6220991

When a newly regenerated kidney is transplanted, the patient's original kidney is preserved and the urinary tract is bound, and the generated urine flows back into the regenerated kidney, inducing hydronephrosis. Therefore, it was necessary to remove the kidney from the patient, and further improvement was required for the practical application of the developed transplant material.

Therefore, an object of the present invention is to provide a transplant material capable of sustaining renal function for a long period of time even if the patient's original kidney is preserved, and a stent used therefor.

The present invention includes the following aspects.
[1] A transplant material comprising a kidney primordium, a bladder bound to the kidney primordium, and a stent that can be deformed into a divergent shape. A transplant material held by the inflated stent and used for transplanting so that the surface in contact with the host's ureter is connected in an expanded state.
[2] The kidney primordium and the bladder are independently composed of substantially autologous or allogeneic cells with respect to host cells, or substantially autologous or allogeneic with respect to host cells. The transplant material according to [1], which is derived from a genetically modified non-human animal that can be replaced with home cells.
[3] The transplant material according to [1] or [2], wherein the joint between the bladder and the ureter is an end-side anastomosis.
[4] The transplant material according to [2] or [3], wherein the genetically modified non-human animal is conditionally provided with a system for removing at least the renal primordium and / or at least a part of the bladder. ..
[5] The transplant material according to any one of [1] to [4], wherein the host is a human.
[6] The transplant material according to any one of [1] to [4], wherein the host is a cat.
[7] The transplant material according to any one of [2] to [6], wherein the non-human animal is a pig.
[8] The transplant material according to any one of [2] to [6], wherein the non-human animal is a mouse.
[9] A stent that can be deformed into a divergent shape and is used for transplanting the transplant material according to any one of [1] to [8] to a host.

According to the present invention, it is possible to provide a transplant material capable of sustaining renal function for a long period of time even while preserving the kidney originally possessed by the patient, and a stent used therefor.

It is a figure which shows an example of the transplant material of this embodiment. It is a figure which showed an example in the living body of the host which transplanted the transplant material. It is a photograph of Experimental Example 1. It is a photograph of Experimental Example 1. It is a photograph of Experimental Example 1. It is a photograph of Experimental Example 1. It is a photograph of Experimental Example 1. It is a photograph of Experimental Example 2.

The present invention is, in one embodiment, a transplant material comprising a kidney primordium, a bladder bound to the kidney primordium, and a stent that can be deformed into a divergent shape, wherein the bladder is inserted therein. After that, a transplant material is provided which is held by the stent which is expanded in a divergent shape and is used for transplanting so that the surface in contact with the ureter of the host is connected in an expanded state.

≪Kidney primordium≫
As shown in FIG. 1, the renal primordium 110 refers to the kidney in the fetal period, and in mammals, it corresponds to the posterior kidney. In anatomical terms, a bird, etc., whose rectum, reproductive organs, and urinary tract system also serve as one excretion port is called a cloaca. The inventors named this fetal bladder-ureter-postrenal tissue cloaca graft. When the renal primordium is transplanted into the abdominal cavity of an animal, blood vessels invade the cloaca from the host, continue to grow, and produce urine.

In the transplant material of the present embodiment, the kidney primordium and the bladder are independently composed of substantially autologous or allogeneic cells to the host cells or substantially autologous to the host cells. Alternatively, it is preferably derived from a genetically modified non-human animal that can be replaced by allogeneic cells.

Hosts to which the transplant material of the present embodiment is transplanted include humans, monkeys, cows, sheep, pigs, goats, horses (particularly horse racing horses), dogs, cats, rabbits, hamsters, guinea pigs, rats, mice and the like. ..

≪Kidney primordium consisting of substantially autologous or allogeneic cells relative to host cells≫
As a method for producing a renal primordia consisting of substantially autologous or allogeneic cells with respect to host cells, mesenchymal stem cells or renal stem cells are injected into the donor's renal development site in the fetus. A method of inducing differentiation into the renal lineage can be mentioned.

Examples of the injected cells include mammalian-derived mesenchymal stem cells, pluripotent stem cell-derived kidney stem cells such as iPS cells, and the like.

The mesenchymal stem cells or kidney stem cells are preferably mesenchymal stem cells or kidney stem cells derived from the host to be transplanted.
Examples of the kidney stem cells include host-derived mesenchymal stem cells (MSCs), kidney stem cells induced to differentiate from pluripotent stem cells such as iPS cells and ES cells, and the like. Kidney stem cells may be cells derived from the patient or the patient, or may be allogeneic cells in which rejection in the patient or the patient is suppressed.

The kidney stem cells are preferably cells derived from the patient or patient to which the kidney after production is transplanted. Examples thereof include kidney stem cells that have been induced to differentiate from mesenchymal stem cells that have been separated from bone marrow, adipose tissue, blood or umbilical cord blood. The preparative method may be a general surgical medical method. Optimal conditions are selected for the separated cells, and the cells are preferably cultured by subculturing 2 to 5 cells. Further, for the purpose of continuing the culture while suppressing the transformation of the mesenchymal stem cells, the culture may be carried out using a medium kit for human mesenchymal stem cells manufactured by Cambrex BioScience.

If desired, a desired gene may be introduced into mesenchymal stem cells or kidney stem cells using adenovirus, retrovirus, or the like. For example, a gene may be introduced to express a glial cellline-developed neurotrophic factor-GDNF for the purpose of assisting kidney formation. This is because the mesenchymal tissue just before kidney formation becomes GDNF-expressing and completes the first important step of kidney development by attracting ureteral buds expressing its receptor, c-ret. Because it makes you. The inventors have confirmed that this transformation increases the formation rate of injected stem cell-derived kidneys from 5.0 ± 4.2% to 29.8 ± 9.2%.

Targets for transplanting mesenchymal stem cells or kidney stem cells include embryos or fetuses in a pregnant mammalian host, or embryos or fetuses isolated from a pregnant mammalian host. By injecting mesenchymal stem cells or kidney stem cells into these subjects, kidneys can be formed in utero.
Examples of the host (donor) include ungulates such as cows, sheep, pigs, goats, and horses; rodents such as hamsters, guinea pigs, rats, and mice.

As a method of transplantation, for example, kidney stem cells may be injected using an injection needle or the like. When the host is a human, the number of human kidney stem cells to be transplanted is preferably, ^{for example, about 1 × 10 3} to 1 × 10 ^6.
It is also possible to directly transplant mesenchymal stem cells into an embryo in a large pregnant mammal such as a pig by a transuterine approach, and continue the growth in the living body as it is to grow into a kidney for transplantation. In addition, the following steps of "whole embryo culture" or "organ culture" may be added.

The prepared mesenchymal stem cells or kidney stem cells are transplanted into a fetus isolated from a pregnant mammalian host (uterine), and then embryos are used in vitro until the initial stage of kidney formation for transplantation is completed. It may be matured in the body and then further cultured in organ culture. In addition, the kidney for transplantation may be transplanted into the omentum of mammals including humans.

The timing of transplantation of the prepared mesenchymal stem cells or renal stem cells into the fetus can be appropriately adjusted. In experiments with rats, 9-12 days of fetal life, eg 10-12 days, eg 10-11.5 days were preferred. Similar stage embryos can be preferably used for large mammalian pigs and the like. In addition, by selecting the conditions, embryos in the stages before and after the above can also be used. Importantly, the host's immune system is still in the stage of immune tolerance, at least at the time of transplantation.

The host immune system is not fully developed at this stage of whole embryo culture. Therefore, it is tolerant of heterologous cells. The endogenous growth system of an immune-protected heterologous host is used to generate autologous organs from autologous mesenchymal stem cells.

By setting the site for transplantation of mesenchymal stem cells or kidney stem cells into the fetus as a site corresponding to kidney development, an organ for transplantation can be suitably prepared. Therefore, it is necessary to perform the transplantation at a time when it can be confirmed that the site corresponds to the development of the kidney, but it is preferable that the transplantation is in a germinated state before the bud cells of the kidney start to develop. For example, by transplanting mesenchymal stem cells or kidney stem cells into posterior nephrogenic mesenchymal cells, it is possible to differentiate into mesandium cells, ureteral epithelial cells, and glomerular epithelial cells, and mesenchymal stem cells or kidney stem cells are intermediate. By transplanting into embryos, it can be differentiated into ureteral bud-derived collecting tubes and ureters.

In the present invention, whole embryo culture is performed when mesenchymal stem cells or renal stem cells are transplanted into a fetus isolated from a pregnant mammalian host (uterus). The uterus is separated from the mother's body, and the fetus is separated from the outer membrane layer including the uterine wall, decidua, and Reichert's membrane. Whole embryo culture can be performed by culturing in-house or the like.

More specifically, the uterus is removed from the mother's body under anesthesia using, for example, a stereomicroscope. Rat embryos 9-12 days, eg 10-12 days, eg 10-11.5 days, eg 11.5 days of embryo are separated from the adventitial layer, including the uterine wall, decidua, and Reichert's membrane. In addition, the yolk sac and amniotic membrane are opened for injection of mesenchymal or renal stem cells, leaving the chorionic allantois placenta intact. Embryos for which successful injection of mesenchymal stem cells or kidney stem cells has been confirmed are cultured in a culture bottle or the like in which 3 mL of a culture medium (dextrose, penicillin G, streptomycin, and amphotericin B) is added to the centrifuged rat serum.

The culture bottle is rotated using, for example, an incubator (model number "RKI10-0310", Ikemoto) or the like. The culturing time may be, for example, 12 hours to 60 hours, for example 24 hours to 48 hours, for example 48 hours. After a certain culture time, evaluate morphologically and functionally to confirm the organ prototype of the kidney for transplantation. The organ prototype may be separated from the fetus and the following organ culture may be performed.

In the present invention, the organ culture is as follows. First, the organ prototype is placed on a filter, and a medium such as DMEM is added to the culture dish under the filter. Then, the culture dish is cultured in a 5% CO ₂ incubator. The culturing time may be, for example, 12 hours to 168 hours, for example, 18 hours to 72 hours, for example, 24 hours to 48 hours, for example, 24 hours. For example, it may be transplanted to the omentum at about 24 hours. The culture temperature may be, for example, 20 to 45 ° C, for example 25 to 40 ° C, for example 37 ° C.

In the present invention, the relay culture means that the whole embryo culture is carried out for 2 hours to 60 hours, and then the organ culture is carried out for 12 hours to 168 hours. Further, after organ culture, it may be transplanted to a host omentum or the like.

The size of the organ is preferably homologous to the organ inherent in the host animal. Therefore, for example, in order to form a kidney capable of exerting a sufficient function in humans, it is preferable that the host is a mammal having a size close to that of a human organ. However, it is not necessary to have the same size at all, and if the kidney has one tenth of the function of the whole, sufficient dialysis can be performed and the life can be sufficiently maintained. For this reason, the optimal host is the pig, and the size of the organ of the miniature pig is judged to be sufficient. In the case of pigs, the above-mentioned whole embryo culture is difficult, so mesenchymal stem cells or kidney stem cells are injected into the renal development site of the fetus by an endometrial operation to prepare an organ for transplantation.

The renal primordium produced as described above is separated from the host and donated to the host after the function is confirmed. After transplantation, the organ for transplantation continues to grow in vivo, and the formation of a cloned kidney that exerts renal function is completed.
The time to remove this renal primordium from the donor is preferably immediately before the vascular invasion period.

The excised renal primordium is then transplanted into the abdominal cavity of the host. The transplantation site is not limited to the abdominal cavity, and examples thereof include a omentum and a para-aorta. The greater omentum is the peritoneum that hangs down like an apron from the underside of the stomach.

For example, transplantation of renal primordium into the omentum, para-aorta, etc. of mammals including humans can be performed by a usual surgical prescription or the like. For example, a method of pinching the kidney primordium to be transplanted with a sharp tweezers, making a slight cut on the surface of the adipose tissue of the omentum with the tip of the tweezers, and embedding the tissue in the cut can be mentioned. It can also be transplanted into the omentum, para-aorta, etc. using an endoscope.

Mesenchymal stem cells or cells derived from kidney stem cells and cells derived from the host animal coexist in the formed kidney. Mixed host-derived cells can cause immune rejection when the kidney is transplanted into the host. Therefore, it is necessary to thoroughly remove host-derived cells after kidney formation.

In order to prevent host-derived antigenic substances from being mixed into the formed kidney, for example, a method of preparing a host animal capable of inducing programmed cell death in a regulated manner and preparing a kidney using this animal as a host. And so on. Mesenchymal stem cells or kidney stem cells are transplanted to the site of the host animal embryo to prepare a kidney, and then cell death is induced in a host cell-specific manner to complete the host-derived cells before transplantation to the host. Can be removed.

For example, iPS cells prepared from the patient's own cells can be differentiated to prepare mesenchymal stem cells and kidney stem cells, and the above method can be applied to prepare regenerated kidneys derived from the patient's own cells. it can.

≪Genetically modified non-human animal-derived kidney primordium that can be substantially replaced by autologous or allogeneic cells with respect to host cells≫
In the process of producing a kidney primordium, it is preferable to use a genetically modified non-human animal as a host (donor) in order to improve the colonization rate of transplanted cells at the fetal kidney development site. Such genetically modified non-human animals may conditionally be equipped with a system that removes at least a part of the renal primordium and / or bladder, such as the above-mentioned host animal capable of inducing programmed cell death in a regulated manner. preferable.

Such a system includes, for example, an iDTR non-human animal expressing a diphtheria toxin receptor (DTR) in a Cre recombinase activity-dependent manner, and a Six2-Cre non-human having a Cre recombinase gene introduced downstream of the Six2 promoter. Examples include a system in which diphtheria toxin is brought into contact with the posterior renal tissue of the resulting offspring by mating with an animal.

iDTR non-human animals have a transcription arrest sequence sandwiched between two loxP sequences upstream of the gene encoding the diphtheria toxin receptor. Therefore, it does not express the diphtheria toxin receptor as it is. However, when the transcription arrest sequence sandwiched between the two loxP sequences is removed by Cre recombinase, the diphtheria toxin receptor is expressed.

For example, since a non-human animal such as a mouse does not have a diphtheria toxin receptor, the cell does not die even if the diphtheria toxin is brought into contact with a cell such as a mouse. However, it is known that when cells such as mice expressing the diphtheria toxin receptor are brought into contact with diphtheria toxin, they die. This phenomenon is used in the above example.

First, when an iDTR non-human animal and a Six2-Cre non-human animal expressing Cre recombinase specifically in the posterior mesenchyme are crossed, a diphtheria toxin receptor is tissue-specifically produced in the posterior renal tissue in the obtained progeny. A non-human animal that expresses appears. Six2 is a transcription factor specifically expressed in the posterior renal mesenchyme.

Subsequently, when the posterior kidney of the non-human animal is brought into contact with the diphtheria toxin, the cells can be specifically killed in the posterior mesenchyme and the renal stem cells in the posterior mesenchyme can be specifically removed. That is, the renal development niche can be opened. Here, the renal development niche refers to a renal development region including a ureteral bud and a posterior renal mesenchyme.

Contact with diphtheria toxin may be performed by injecting diphtheria toxin into the fetus of a non-human animal. Alternatively, the posterior kidney may be removed from the fetus of a non-human animal, the removed kidney may be organ-cultured, and diphtheria toxin may be added to the medium. Alternatively, the excised kidney may be transplanted into the para-aortic region or body network of the patient or the patient, and diphtheria toxin may be locally administered in the body of the patient or the patient.

In the above-mentioned example, when a non-human animal in which a gene encoding the Cre-ER protein is further introduced downstream of the ureteral bud-specific promoter is used, the ureteral bud can be specifically removed.
Here, examples of the ureteral bud-specific promoter include a promoter of cytokeratin 8 and a promoter of HoxB7.

Such mice include, for example, iDTR non-human animals having the genotype of ^{Six2-Cretg / wt} cytokeratin 8-Cre-ER ^{tg / wt , Six2-Cretg / wt} HoxB7-Cre-ER ^{tg / wt} . Included are iDTR non-human animals with genotype. Here, "tg" indicates that it is transgenic, and "wt" indicates that it is a wild type.

The Cre-ER protein is a fusion protein of Cre recombinase and mutant estrogen receptor. The non-human animals express Cre-ER in a promoter-dependent manner of cytokeratin 8 or Hox B7, which is a marker of ureteral buds.

The Cre-ER protein is normally present in the cytoplasm, but when it binds to the estrogen derivative tamoxifen, it translocates into the nucleus and undergoes recombination with respect to the loxP sequence. Utilizing this, it is possible to adjust the working time of the Cre-loxP system in a tamoxifen-dependent manner. Therefore, even if Cre-ER and Cre are expressed at the same time, the expression of Cre-ER activity can be controlled by whether or not tamoxifen is administered. Here, instead of Cre-ER, Cre-ERT, Cre-ERT2, etc., which are variants of Cre-ER, may be used.

In addition to, for example, a construct that expresses a diphtheria toxin receptor (DTR) in a Cre recombinase activity-dependent manner, and a construct in which the Cre recombinase gene is linked downstream of a posterior mesenchymal promoter, urinary tract buds are further added. By using a genetically modified non-human animal having a construct in which the Cre recombinase gene is linked downstream of a specific promoter, the kidney primordia derived from the genetically modified non-human animal can be substantially autologous or allogeneic to the host cell. Can be replaced with home cells.

Examples of the method for substituting the renal primordium derived from the genetically modified non-human animal with substantially autologous or allogeneic cells for the host cells include the following methods. First, the diphtheria toxin is brought into contact with the posterior kidney to specifically remove the renal stem cells of the posterior mesenchyme. After or at the same time as the removal of renal stem cells in the posterior mesenchyme, substantially autologous or allogeneic renal stem cells are transplanted into the host cells, and the transplanted cells are differentiated and matured to release a part of the renal primordia. To form. The ureteral buds are then specifically removed by contacting tamoxifen with the posterior kidney. After or at the same time as ureteral bud removal, substantially autologous or allogeneic allogeneic kidney stem cells are transplanted into host cells, and the transplanted cells are differentiated and matured to form part of the renal primordium. By such a method, the kidney primordium derived from a genetically modified non-human animal is replaced with substantially autologous or allogeneic cells relative to the host cells.

Here, "substantially" means that when a kidney is produced using the renal development niche of a non-human animal, the cells of the transplanted host are substantially other than autologous or allogeneic kidney stem cells. It means that it does not exclude the contamination of cells. In other words, the regenerated glomerules and tubules preferably consist of 70% by mass or more of the transplanted cells, more preferably 80% by mass or more of the transplanted cells, and 90% by mass or more of the transplanted cells. It is even more preferably composed of 95% by mass or more of the transplanted cells, and 99% by mass or more of the transplanted cells is particularly preferable.

In addition, the system for removing at least a part of the renal primordium may be a configuration in which cells are killed by a system other than diphtheria toxin or diphtheria toxin receptor. For example, a system that expresses a herpesvirus-derived thymidine kinase gene (HSV-TK) that induces apoptosis by administration of ganciclovir downstream of a tissue-specific promoter can be mentioned. Cells expressing HSV-TK are induced to die under ganciclovir administration.

Alternatively, a system that induces apoptosis by dimerizing Caspase3, Caspase8, Caspase9, etc. by administration of AP20187 may be used.

A similar method can also be used for the bladder.

Examples of non-human animals in the above-mentioned genetically modified non-human animals include ungulates such as cows, sheep, pigs, goats and horses; rodents such as hamsters, guinea pigs, rats and mice.

≪Transplant material≫
FIG. 1 is a diagram showing an example of a transplant material of the present embodiment. As shown in FIG. 1, the kidney primordium 110 and the bladder 120 bound to the kidney primordium 110 enter the host ureter 140 via a stent 130 inserted inside the bladder 120 and inflated into a divergent shape. Be connected. When the stent 130 expands in a divergent shape, the bladder 120 also expands in a divergent shape and is connected to the host ureter 140 with the surface in contact with the host ureter 140 expanded.

This implant material grows in vivo in the host's body and becomes urine-producing. The produced urine is transferred to the host's bladder via the host's ureter 140 and excreted.

The transplant destination of the transplant material in the host is not particularly limited as long as the bladder 120 and the host ureter 140 can be connected, but the vicinity of the host omentum, para-aorta, and splenic artery is preferable. FIG. 2 is a diagram showing an example in vivo of a host transplanted with a transplant material. In the transplant material 100, the kidney primordium 110 side is transplanted into the para-aorta 150 of the host and connected to the ureter 140 of the host via a stent 130.

In the transplant material 100, the animal from which the kidney primordium 110 and the bladder 120 are derived may be the same species as the host or a different species. For example, a porcine-derived transplant material can be transplanted into a human, a cat, or the like to form an organ structure. This is one of the options in the current situation where there is a shortage of transplantable kidneys.

In the transplant material 100, the kidney primordium 110 and the bladder 120 may be derived from a genetically modified non-human animal of a species different from the transplant target. For example, in the transplant material 100, the kidney primordium 110 and the bladder 120 may be derived from genetically modified pigs with reduced rejection when transplanted into humans or cats.

When the transplant material 100 is transplanted into a human, the kidney primordium 110 and the bladder 120 are preferably composed of substantially human cells, and most preferably composed of the cells of the patient himself / herself. Here, "substantially" means that when a transplant material is produced using the body of an animal or the like, contamination with cells other than humans is not excluded. In other words, 90% or more of the cells constituting the transplant material are preferably human cells, 95% or more are more preferably human cells, and 99% or more are particularly preferably human cells.
The bladder 120 may be of donor origin. For example, it may be derived from a pig, and the bladder can be removed and replaced with host ureteral epithelial cells using the system for removing bladder constituent cells as described above.

≪Stent≫
The present invention provides, in one embodiment, a stent that is deformable into a divergent shape and is used to implant the implant material into a host.

The stent in the present embodiment is not particularly limited as long as it is used for surgery and can be deformed into a divergent shape as shown in FIG. For example, a stent used for a narrowed portion of a coronary artery.
For example, as a method of using the stent of this embodiment, the following method can be mentioned. The bladder of the transplant material transplanted to the host is cut, a catheter having a balloon with a stent at the tip is inserted into the bladder, and the stent is spread in a divergent shape. Remove the balloon catheter, leaving the widened stent. As a result, the lumen of the bladder remains open and does not cause stenosis.

≪Treatment method≫
In one embodiment, the invention independently comprises substantially autologous or allogeneic cells relative to the host cell, or substantially autologous or allogeneic cells relative to the host cell. The step (a) of transplanting a renal primordia and a bladder bound to the renal primordia, which is derived from a genetically modified non-human animal that can be replaced with, into the body of a patient or a patient, and a predetermined period after the transplantation, A stent that can be deformed into a divergent shape is inserted inside, the stent is inflated into a divergent shape, and the bladder is connected to the host ureter with the surface of the bladder in contact with the host ureter expanded. Provided is a method for transplanting a kidney, comprising the step (b).
The host is a patient or a patient, and examples of the patient or a patient include humans and cats.

It can be said that the method of the present embodiment is a method for treating renal disease. As the transplant material, the above-mentioned materials can be used.

By leaving the transplanted material transplanted in the above step (a) for a predetermined period of time, the transplanted material grows and starts urine production. The predetermined period may be a period before the transplant material grows sufficiently and hydronephrosis occurs. The predetermined period may be appropriately adjusted according to the symptoms of the patient or the patient, and may be, for example, 1 to 10 weeks.

Subsequently, step (b) is carried out after a predetermined period from the transplantation. In step (b), the bladder of the implant material is cut, stunted and inflated to form a divergent shape, and then the bladder is connected to the ureter of the patient or patient. Examples of the connection method include end-to-end anastomosis, end-side anastomosis, side-to-side anastomosis, and side-end anastomosis, and end-to-end anastomosis is preferable.

By connecting the bladder of the transplant material and the bladder of the patient or the patient via the ureter of the patient or the patient, the urine produced by the transplant material can be excreted into the bladder of the patient or the patient. More specifically, the urine produced by the kidney of the transplant material is excreted to the bladder of the transplant material, and then from the bladder of the transplant material to the bladder of the patient or patient through the ureter of the patient or patient.

In step (b), when the bladder of the transplant material is derived from a genetically modified non-human animal, the bladder derived from the non-human animal is removed by using the above-mentioned system for removing bladder constituent cells conditionally. According to such removal, the mucous membrane of the bladder disappears and fibrosis is promoted, but in the present invention, since the stent is provided, the lumen of the bladder remains open and stenosis does not occur. Eventually, the bladder of the implant material will be replaced by that of the host, as the host's ureteral epithelium will grow to compensate for the bladder deletion as the bladder of the implant material is removed.

According to the method of the present embodiment, the urine produced by the kidney of the transplant material can be excreted into the bladder of the patient or the patient while preserving the kidney of the patient or the patient.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

[Experimental Example 1]
Five fetuses were taken out from one pregnant micromini pig, and a cloakagraft (kidney primordium graft with bladder) was prepared under a microscope. Enhancing Survival of Human Hepatocytes by Neonatal Thymectomy and Partial Hepatectomy in Micro-miniature Pigs. Hsu HC, Enosawa S, Yamazaki T, Tohyama S, Fujita J, Fukuda K, Kobayashi E Two males of Transplant Proc. 2017 Jan --Feb; 49 (1): 153-158.) Were transplanted with cloakagrafts (see Figure 4). For the host's right kidney, a 50% renal infarction was made, the splenectomy was performed, and a gastrostomy tube was inserted. Postoperatively, according to the immunosuppressive protocol developed by the inventors (Development of an immunodeficient pig model allowing long-term accommodation of artificial human vascular tubes. Itoh M, Mukae Y, Kitsuka T, Arai K, Nakamura A, Uchihashi K, Toda S, Matsubayashi K, Oyama JI, Node K, Kami D, Gojo S, Morita S, Nishida T, Nakayama K, Kobayashi E. Nat Commun. 2019 May 21; 10 (1): 2244.), Host Managed.

After one month of immunosuppression, the host transplanted with cloaca graft was restarted and the growth of cloaca graft was confirmed (see FIG. 5). The fetal kidney, which had a diameter of 2 mm or less, grew to about 1 cm, and a bladder was confirmed. The transplantation of the cloaca graft was previously developed by the inventors (Urine excretion strategy for stem cell-generated embryonic kidneys. Yokote S, Matsunari H, Iwai S, Yamanaka S, Uchikura A, Fujimoto E, Matsumoto K, Nagashima H, Kobayashi E, Yokoo T. Proc Natl Acad Sci US A. 2015 Oct 20; 112 (42): 12980-5.) However, this time, we examined whether urinary tract reconstruction could be performed by end-side anastomosis without removing the kidney of the host (see FIG. 6).
As shown in FIG. 6, the developed cloaca bladder was subjected to end-to-end anastomosis and end-to-end anastomosis, respectively.

After the anastomosis, immunosuppression was performed for another month, and the growth of the cloaca graft was confirmed (see FIG. 7). The cloaca graft of the bladder / ureteral end-side anastomosis was further developed with blood vessels entering directly from the host aorta.

[Experimental Example 2]
A total hysterectomy was performed from a pregnant micromini pig, and a fetus in late pregnancy was taken out. The cloaca graft (kidney primordium graft with bladder) used was one in which the development of the bladder could be confirmed at this point. First, the thymus and splenectomy of the mother pig from which the fetus was taken out were performed. The bladder incision edge of the cloaca graft and the stent were then fixed with 6-0 microthreads and end-to-end anastomosis with the host ureter (see FIG. 8). The urinary tract could be reconstructed without removing the host's kidneys.

According to the present invention, it is possible to provide a transplant material capable of sustaining renal function for a long period of time even while preserving the kidney originally possessed by the patient, and a stent used therefor.

100 ... Transplant material, 110 ... Kidney primordia, 120 ... Bladder, 130 ... Stent, 140 ... Host ureter, 150 ... Host para-aorta, 160 ... Host kidney.

Claims (9)

A transplant material comprising a kidney primordium, a bladder bound to the kidney primordium, and a stent that can be deformed into a divergent shape.
A transplant material used for implanting the bladder, which is inserted into the bladder, is held by the stent which is expanded in a divergent shape, and is connected so that the surface in contact with the ureter of the host is connected in an expanded state. The kidney primordium and the bladder independently consist of substantially autologous or allogeneic cells relative to host cells, or substantially autologous or allogeneic cells relative to host cells. The transplant material according to claim 1, which is derived from a genetically modified non-human animal that can be substituted with. The implant material according to claim 1 or 2, wherein the joint between the bladder and the ureter is an end-to-side anastomosis. The transplant material according to claim 2 or 3, wherein the genetically modified non-human animal conditionally comprises a system for removing at least the kidney primordium and / or at least a part of the bladder. The transplant material according to any one of claims 1 to 4, wherein the host is a human. The transplant material according to any one of claims 1 to 4, wherein the host is a cat. The transplant material according to any one of claims 2 to 6, wherein the non-human animal is a pig. The transplant material according to any one of claims 2 to 6, wherein the non-human animal is a mouse. A stent that can be deformed into a divergent shape, which is used for transplanting the transplant material according to any one of claims 1 to 8 to a host.

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