Challenges for Production of Human Transplantable Organ Grafts

Abstract

The described research methods explain how you could generate a three-dimensional kidney, based on recent research results. The first method is to fabricate human organs in a pig body. The second is to transplant the so-called “organ bud” into a patient’s body for further development. The third method is to regenerate organs by filling cells into the cytoskeleton as a scaffold. Research for the in vitro fabrication of organ buds has been elaborately accelerated. The organ bud transplantation has been confronted with issues of continuity with the original organs, so the development of technology for achieving continuity between a transplanted organ bud and the existing organs is progressing well. The “organ fabrication” methodology, whereby cells are placed into completely decellularized organs, is supported by recent research results using pig organs taking the size of humans into consideration.

INTRODUCTION

End-stage organ failure treatments currently depend on organ transplantation. However, there is a serious shortage in the number of transplantable organs. For example, end-stage renal failure treatments consist of hemodialysis, peritoneal dialysis, and kidney transplantation. Nevertheless, in Japan, the reliance on hemodialysis has been very high compared to that of other countries worldwide, and the issue has been highlighted from a medical economy point of view. Furthermore, kidney transplantation still relies too much on living donors, resulting in an insufficient situation1. The author has previously reported on transplantation tourism and risk as a 2006 special research project of the Ministry of Health, Labor and Welfare and has referred to the unethical situation for kidney transplantation in overseas countries in which Japanese patients suffering from renal failures have been involved2. These academic activities centered by The Japan Society for Transplantation in collaboration with The Transplantation Society and International Society of Nephrology played an important role in the 2008 Declaration of Istanbul, which was regarded as an international declaration for organ trafficking and transplantation tourism. Consequently, this had great influence on the reform bill for organ transplantation3. Efforts have been made to expand transplantation from a brain-dead donor and to increase the safety of living donors, but the number of kidney transplantations has not been increasing due to a decrease in kidney donors (kidney transplantation after cardiac arrest).

Reconsidering the situation of an extremely small number of kidney transplantations from brain-dead donors, society should, hand in hand, take action after deep consideration, and simultaneously we should bear in mind the efforts made by our senior researchers for translational research in Japan. Under the situation that organ transplants have not been legal, research made to expand the donor pool by Takahashi and Saito4 and Matsuno et al.5 have had great impact globally on kidney transplantation and contributed to clinical trials.

This review depicts strategies of how we can expand the optimization of organs from non-heart-beating donors (NHBDs) using a pragmatic point of view. The challenging research to fabricate organs is also described in case the revivification of organs turns out to be a dead end.

THE CHALLENGE TO OPTIMIZE THE USAGE OF ORGANS FROM NHBDs

On the basis of a series of studies, the author has been promoting the methodology to resuscitate organs in case of kidney transplantation from cardiac arrest by breaking through the existing concept of organ preservation (Fig. 1)6. The donor organs from cardiac arrest tend to stay under extreme hypoxia and have decreased internal adenosine triphosphate (ATP) levels. The current low-temperature preservation method is effective only for reducing the speed of worsening viability after resection but never turns out to improve7. In recent years, it has become a tendency to try to keep the organs in cardiac arrest over 20°C8. The author’s group has demonstrated using a rat model that trehalose-containing extracellular preservation liquid is effective for keeping organs between 20°C and 22°C9 and has also proven that oxygen carriers such as hemoglobin are indispensable due to the fact that oxygenation through bubbling is noxious because it generates active oxygen10. Through noninvasive diagnostic imaging by magnetic resonance imaging (MRI), it has been shown that postoperative prognosis after organ transplantation such as kidney as a marginal transplantation candidate has become viable11. Furthermore, it has been reported that the function of transplanted marginal kidney would be improved by adding mesenchymal stem cells (MSCs) as an optional transplantation12. In addition, the research has been developed to the extent that newly fabricated human kidney cells are able to repopulate the dead kidney extracellular matrix through the perfusion decellularization method6.

Figure 1.

Proposed protocol to accelerate kidney transplantation from donors with cardiac arrest (reproduced with permission from Tokyo Igakusha Ltd., publishers of Kobayashi6). First, the kidney is resected from a non-heart-beating donor (NHBD) and perfused in organ preservation solution at 22°C, though perhaps it might be better to call it organ resuscitation solution. The noninvasive evaluation is performed by magnetic resonance imaging (MRI) to judge whether the resuscitated kidney is transplantable. This leads to the following three operative decisions. (1) If the tissue is fully resuscitated, then transplantation is performed. (2) In case of insufficient resuscitation, regeneration-inducing stem cells such as mesenchymal stem cells (MSCs) are administrated to the whole body after transplantation (Tx). (3) In case it is judged that the resected kidney is not suitable for transplantation, it is decellularized and regenerated. If such a protocol becomes generally accepted, goodwill extended by the donor is fully optimized.

THREE WAYS TO FABRICATE TRANSPLANTABLE ORGANS

Research methods to fabricate organs, such as the transplantable kidney with vascular structure, based on our frontline research can be categorized into three general procedures. The first is “so-to-speak” to generate organs through the use of a whole animal body. The second is to transplant the organ bud and grow it in the patient’s body. The third is to regenerate organs by replenishing cells on a decellularized cytoskeleton as scaffolds. They are explained in detail below.

Nurture Human Organs in Xenogeneic Animals (Such as Pigs)

This method is frequently known as “in vivo bioreactor” (animal factory). It means that human stem cells are injected into xenogeneic animal fetuses before their immune function is completed in order to utilize the growth process of fetuses for human stem cell differentiation into organs.

The first trial was performed by Mackenzie and Flake using human MSCs that were injected into sheep embryos in utero13. This method of injecting human MSCs into embryos in vitro to generate an organoid of kidney was directly proven by Yokoo’s team14. In recent years, the team of Nakauchi explored organogenesis using small animals through the injection of induced pluripotent stem/embryonic stem cells (iPSCs/ESCs) into the allogeneic mouse fertilized egg. They used the blastocyst replacement method with the knockout of genes relating to kidney development [in this case spalt-like transcription factor 1 (Sall 1)], which could be replaced by the injected cells leading to organogenesis15. If used clinically, then this method would need to solve ethical issues in Japan because it would end up generating a xenogeneic embryo between an animal and a human. The overall issues with regard to in vivo bioreactors are directly connected to fundamental ethical issues in that we need to sacrifice pigs and other experimental animals in order to generate human organs. However, there is clearly future potential in that we can artificially generate the vital environment to grow organs by pushing forward with the current research.

Nurture Human Organ Bud in Patient’s Body

There is a method called “organ bud transplantation,” meaning that an in vitro generated human organ bud is transplanted into the patient’s body for further growth. Recently, two superb research results have been reported16,17. The team of Osafune generated human distal ureter from the cocultivation of intermediate mesoderm cells through the differentiation of embryo kidney cells of mouse and human iPSCs16. The team of Sakaguchi has generated somatic stem cells from human iPSCs and also kidney precursor cells17, with which they succeeded in generating human glomerulus and renal tubule through contact cultivation with the embryo spiral code of a mouse18. Both studies make clear the mechanism of kidney development for the purpose of generating the three-dimensional (3D) structure of the kidney. Just recently, a team in Australia has succeeded in cultivating the most renal-related cells as an organoid in vitro from human iPSCs19.

These attempts to grow renal organ buds in vitro as an organoid and transplant them into a patient’s body for further growth shine a spotlight on organ bud transplantation. In 2006, Takeda’s group experimented with kidney and pancreas organ bud from embryo pigs into rats20. The author and the team of Yokoo have been researching human chimerization by injecting MSCs as human somatic cells into animal kidney buds. An immunosuppression is needed temporarily to grow pig’s kidney bud with human MSCs inside a patient’s body. It has been shown that it is possible to generate complete human chimera tissues if the host animal tissues were induced to be eliminated at the stage that human stem cells are differentiated to kidney cells within the xenotissue21. Nevertheless, the limit of this method is that the route to drain secretions such as bile and urine outside the body is not naturally made. The organ bud transplanted into the patients should grow in continuation with the organs of the recipients.

Recently, we have been able to overcome the issue of this continuity by the cloaca transplantation method22. That is, cloaca as an anlage of the kidney connected with the urinary tract as a continuation is transplanted into the recipient for growth and the connection of the cloaca urinary bladder with the urinary duct of the recipient (Fig. 2).

Figure 2.

The newly regenerated kidney transplantation methodology we have been proposing (reproduced with permission from National Academy of Sciences, publishers of Yokote et al.22). (A) Inside the body of a patient, kidney and urinary tract networks are developed through the transplantation of the cloaca, which is the integration of the kidney bud, urinary tract, and bladder. By connecting a grown urinary bladder with the urinary tract, it becomes possible to maintain regenerated kidney functions. (B) Currently, kidney bud regenerative transplantation has resulted in hydronephrosis due to the fact that the renal pelvis part is swollen by urine.

Refilling Human Cells to the Xenogeneic Organs Decellularized Extracellular Matrix as Scaffolds

In recent years, the method of utilizing 3D vascular plexus as an extracellular matrix through the washout process of cells from animal organs (decellularization) to be used as scaffolds for refilling cells has been astonishingly developed. The extracellular matrix consists of a line of epithelial cells and connective tissue of stroma. Washing out the cells through decellularization leaves the microcirculation environment intact to some extent. In recent years, the research field with rat models has been making great progress23,24. The team of Ross reported in detail the use of decellularized rat kidney to explore the differentiation process in the kidney of cells after the injection of mouse ESC antegrade from the kidney veins and retrograde from the urinary duct23. In addition, a group at Massachusetts General Hospital in the US has been trying to decellularize rat, pig, and human kidneys. In the case of the rat, after cultivating through reinjection of cells to the decellularized graft, it was reported that sympatric kidney transplantation resulted in the emergence of urine24.

The author’s team has been trying to decellularize pig organs for the purpose of human-size organ experiments. Various organs have been resected from experimental pigs and frozen at −80°C after setting up a perfusion circuit. The frozen organs were defrosted at room temperature, and the surfactant was circulated for decellularization. This project dubbed “research for scaffold” has been pushed forward by utilizing liver, kidney, lung, heart, spleen, and small intestine of experimental pigs.

Regarding pilot experiments for kidney decellularization, after confirming partial adhesion to the decellularized graft through the filling of pig iPSCs, MSCs, and vascular endothelial cells, the graft was transplanted into another pig with vascular network. After transplantation, the arteries of the transplanted kidney were perfused with heparin. After the operation when blood flow was lowered, the pig was sacrificed and the kidney status has been observed with the follow-up of computed tomography (CT) (Fig. 3). Also concerning experiments for liver decellularization, pig hepatocytes have been cryopreserved and used to fill livers and transplanted for further study. The segmentum of the pig small intestine has been decellularized and adhered to a cardiomyocyte sheet to derive a vascular bed to extend research.

Figure 3.

Transplantation of regenerated kidney based on the pig’s decellularized kidney (reproduced with permission from Tokyo Igakusha Ltd., publishers of Kobayashi6). (A) Change of colors before and after decellularization. (B) Right after transplantation: almost normal color. (C) Computed tomography (CT) image after 1 week: decrease in blood flow is shown by a blue arrow. (D) Comparison with normal kidney (right) versus “nutmeg”-like kidney (left).

Up to now, long-term blood flow has not been acquired. However, it is expected that in addition to the improvement of endothelium stickiness to the decellularized vascular network, the growth of revitalized organs through the anticoagulant method within a short period after the operation is viable. In order to fabricate human-size organs, technologies to provide a large amount of cell cultivation, its cell refilling, and 3D cell cultivation are to be challenged.

CONCLUSIONS

In the 20th century, organ transplantation including kidney transplantation has become an ultimate treatment method to save patients facing death from organ failures and return them to a healthy condition. Nevertheless, this ultimate medical practice always involves human donors. “Fabricating transplantable organs” is therefore indispensable in the future for developing therapy without relying on the availability of donors either from brain death or living donor transplantation. The extremely challenging science to fabricate entire organs (regenerative medicine) requires the centralization of knowledge from various kinds of research fields. In this summary, some of the challenges on clinical application for fabricating entire organs have been described, and the author is continuously pushing forward with these studies as viable mechanisms of organ generation.