Clinical Trials of Stem Cell Therapy in Japan: The Decade of Progress under the National Program

Stem cell therapy is a current world-wide topic in medical science. Various therapies have been approved based on their effectiveness and put into practical use. In Japan, research and development-related stem cell therapy, generally referred to as regenerative medicine, has been led by the government. The national scheme started in 2002, and support for the transition to clinical trials has been accelerating since 2011. Of the initial 18 projects that were accepted in the budget for preclinical research, 15 projects have begun clinical trials so far. These include the transplantation of retinal, cardiac, and dopamine-producing cells differentiated from human induced pluripotent stem (iPS) cells and hepatocyte-like cells differentiated from human embryonic stem (ES) cells. The distinctive feature of the stem cell research in Japan is the use of iPS cells. A national framework was also been set-up to attain the final goal: health insurance coverage. Now, insurance covers cell transplantation therapies for the repair and recovery of damaged skin, articular cartilage, and stroke as well as therapies introduced from abroad, such as allogeneic mesenchymal stem cells for graft-versus-host disease and chimeric antigen receptor-T (CAR-T) cell therapy. To prepare this review, original information was sought from Japanese authentic websites, which are reliable but a little hard to access due to the fact of multiple less-organized databases and the language barrier. Then, each fact was corroborated by citing its English version or publication in international journals as much as possible. This review provides a summary of progress over the past decade under the national program and a state-of-the-art factual view of research activities, government policy, and regulation in Japan for the realization of stem cell therapy.


Introduction
Human whole genome sequencing and the progress in the elucidation of a biological reaction network have resulted in a new scientific term: bioinformatics. Currently, a number of biological events can be explained at the molecular level. The acceleration of research and development in life sciences has led to a global trend: the 21st century is the biotechnology era [1]. In Japan, the Biotechnology Strategy Council, established by the Cabinet Office of the Japanese government issued "Strategies for the Development of Biotechnology" in December 2002 as a vision of the 21st century [2][3][4]. This report proposed a new direction in science and technology, i.e., a shift from electronics to biotechnology. In response to this report, the Project for Realization of Regenerative Medicine was launched in 2003 [5].
Regenerative medicine originates from stem cell biology in embryology. The therapeutic concept is to restore the function of damaged organs and tissues by stem cells. The implantation or mobilization of stem cells shows unique potency not yet seen in existing pharmaceuticals, and the cell itself is being recognized as a new medicinal category. In particular, such therapy is expected to replace organ transplantation, which has a serious long-lasting donor shortage. In research and development, somatic stem cells and embryonic stem (ES) cells preceded and induced pluripotent stem cells (iPS) cells established by Shinya Yamanaka, a Nobel Prize awardee in 2012 [6,7]. iPS cells enable the use of autologous pluripotent stem cells, which are produced from somatic cells, and resolve not only the issue of the immunological rejection but also the ethical concerns of ES cells. The establishment of iPS cells was an epochal achievement of the biotechnology era in Japan.
In 2011, the Highway Program for Realization of Regenerative Medicine began, and it played a major role in the transition to clinical applications. The program is set under the Research Center Network for Realization of Regenerative Medicine, and the support for preclinical studies was enhanced. Of the 18 projects initially selected for the program, 15 projects have announced clinical implementation to date. In this review, the past and current status of clinical trials of stem cell therapy in Japan is discussed focusing on the progress under the national program.

Preclinical Research Projects Aiming at the Transition to Clinical Trials
The Highway Program for Realization of Regenerative Medicine and the Program of the Research Center Network for Realization of Regenerative Medicine recruited 18 scientific projects, 1 foundational platform, and 2 support programs ( Table 1). The objective was to start clinical trials in 3 to 7 years. The scientific projects were categorized by the use of human somatic cells and the use of human iPS and ES cells. The former includes treatments for articular cartilage, corneal endothelium, liver cirrhosis, and intestinal epithelium, totaling five projects. The latter include treatments involving the eye (three projects), central nervous system (two projects), heart (two projects), liver (two projects), articular cartilage (one project), pancreatic islets (one project), and immune system (one project), totaling 13 projects, one of which involves the application of human ES cells and the rest the application of human iPS cells. Supporting programs cover regulation and ethical, legal, and social issues (ELSI), and the two teams organized regular meetings to instruct on key issues for the successful transition to clinical trials in a cross-sectoral manner. Each project has its own originality based on the preceding research with somatic stem cells as well as human ES and iPS cells. Sekiya et al. first determined the superiority of mesenchymal stem cells isolated from synovium in chondrogenesis activity compared to stem cells from bone marrow, periosteum, skeletal muscle, and adipose tissue [8] and confirmed the repairing ability in a rabbit osteochondral defect model in vivo [9]. To obtain cells for transplantation of corneal endothelium, the disfunction of which induces a serious homeostatic imbalance of the anterior hydatoid, a ROCK inhibitor treatment was effective in triggering the cells' proliferation, which was otherwise static [11]. The procedure enabled the novel treatment of cell engraftment for corneal endothelium disorder instead of corneal tissue transplantation [10]. The therapeutic potential of mesenchymal stem cells in the amelioration of cirrhotic liver has been investigated using autologous bone marrow cells [12]. However, it is often hard to harvest enough cells from patients with liver cirrhosis under general anesthesia. Thus, a method to increase the number of cells prior to transplantation was established [13]. In the mesenchymal stem cell therapy of knee joint cartilage, the preloading of magnetic beads with a medical grade helped to position the cells in the most effective place for the treatment [14]. The colonic stem cell-derived organoids showed the integration and repairment of an artificial ulcer in mouse colon, promising clinical application for inflammatory bowel diseases [15].
Most of the projects involving human iPS and ES cells are aimed at the transplantation of manufactured tissue to replace diseased tissue. All of the studies proposed the resolution of unmet medical needs by producing cells and tissues through quality controlled processing. For instance, the target disease of the retinal tissue regeneration was age-related macular degeneration [16,17]. There are a number of patients but still few effective treatments, except for the periodic topical injection of anti-vascular endothelial growth factor (VEGF) antibody to suppress retinal pathogenic thickening. Translocation of the perimacular area of a patient's own retina to the diseased macular area was tried using intraocular microsurgery [36], but it is not conducted at present because of the invasiveness and lack of an obvious effect. Transplantation of stem cell-derived retinal tissue is hoped to be a permanent treatment, especially in order to escape the need for regular injections. In addition, there are also ethical rationales for conducting such clinical trials: (1) the disease is not life-threatening but highly impairs the quality of life; (2) the intraocular region is an immune privilege site; (3) carcinogenesis in the retina is very rare in adults. The project involving cell transplantation for Parkinson's disease proposes to use human iPS cell-derived dopamine-producing cells [21]. A similar treatment was attempted with allogeneic fetal olfactory mucosal cells isolated from aborted fetus [37,38]. The procedure involved serious ethical issues and the effect was disputable. Olfactory cells contain not only dopaminebut also serotonin-secreting cells, which are not effective but rather aggravative toward neurological symptoms. The project aims to procure and transplant purified dopamine-producing cells under well-controlled cell processing manufacturing that is free of ethical concerns. Articular cartilage is commercially available [39] and its therapeutic potential is well known [40]. However, resource is limited because only cartilage from child and juvenile donors is effective. The human iPS cell-derived cartilage has characteristics of early developmental stages and is considered more suitable for cartilage repair [31]. In addition to the above-mentioned projects, clinical trial-oriented research on stem cell therapy for cornea [18,19], heart [20,28], liver [22,29], platelets [25], spinal cord [26], immune system (natural killer T cells) [30], and pancreatic islets [32] was started.
At the time of proposal, the projects using iPS cells were envisioned to use the iPS cells produced from the patient's own somatic cells such as subcutaneous fibroblasts or lymphocytes. Later, the cell source was changed to the iPS cell stock as mentioned below (see Section 3.3) and all clinical trials are to use allogeneic iPS cells except for Masayo Takahashi's first trial and Koji Eto's platelet trial (6 -1 and 11 in Table 2).

Legislation
Along with the increased support in the research budgets, the adjustment of regulations is important for conducting a national scheme. Previously, only an explanatory clinical run of a cell therapy was allowed, under a decision by the head of the institution in accordance with a report by the institutional review board, which was installed based on national ethics guidelines. At that time, the data were not valid for official investigations of new drugs. In 2014, the revised Act on Securing Quality, Efficacy and Safety of Products Including Pharmaceuticals and Medical Devices (Act No.145 of 10 August 1960) was enacted [41] and it included two new categories: regenerative medical products and gene therapy products. Regenerative medical products consist of human somatic stem cell-and human somatic cell-processed products at present, because there are no approved products with cells derived from iPS and ES cells. Characteristically, a system of conditional and time-limited approval was introduced for regenerative medical products. The purpose of this system is to apply the products to patients if they are considered to be effective, since it often takes time to determine the effectiveness of regenerative medicine. Once approval or conditional and time-limited approval is given, the medical treatments with the products are covered by health insurance.
In 2014, the Act on the Safety of Regenerative Medicine (Act No.85 of 27 November 2013) was enforced [42]. According to this law, the Ministry of Health, Labor, and Welfare examines all protocols of clinical research on regenerative medicine that had been regulated under national ethics guidelines before. The pancreatic islet transplantation was first performed under this law and after the data estimation was completed, the procedure was authorized and covered by health insurance in 2021, as introduced by Noguchi [43].

Japan Agency for Medical Research and Development (AMED)
The Japan Agency for Medical Research and Development (AMED) was established in 2015, as the headquarter that play a central role in the promotion of research and development in the medical field and transition to commercialization [44]. The budget for clinical-oriented research and development, which had been allocated independently by the Ministry of Health, Labor and Welfare, the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Economy, Trade, and Industry, is now allocated by the AMED. The AMED also maneuvers the approved research projects for their satisfactory execution.

iPS Cell Stock Project
The advantage of iPS cells in stem cell therapy is the availability of autologous iPS cells, which escape from allogeneic immune rejection. However, the establishment of an iPS cell line is extremely time and cost consuming, especially to establish clinical-grade iPS cell lines that are secured by good manufacturing practice (GMP). Moreover, the acquire-ment of a successful iPS cell line is affected by individual donor differences, including age. Therefore, the Program of the Research Center Network for Realization of Regenerative Medicine shifted to the use of allogeneic iPS cell lines from tailor-made autologous iPS cells. The iPS cell stock project embarked to produce a collection of iPS cell lines from persons whose human leukocyte antigen (HLA) haplotype is homozygous [33][34][35]. As far as HLA-A, HLA-B, and HLA-DR are concerned, the compatibility of those homozygous cells is wider than for heterozygous cells, and 140 homohaplotype lines will cover 90% of the Japanese population [35]. Although those cells are still allogeneic in minor histocompatibility antigens, the rejection reaction is thought to be mild. Currently, GMP-secured homohaplotype iPS cell lines are used in preclinical studies that are in the middle to late stages when clinical trials are within range. Table 2 displays a list of the clinical trials that were derived from the projects shown in Table 1. All projects with somatic stem cells have started, and some results have been published. The project involving the repair of articular cartilage has four subsidiary trials (1 -1 , 1 -2 , 1 -3 , and 1 -4 ) from slightly different perspectives. Likewise, the projects involving corneal and liver regeneration have set four and three protocols, respectively. The clinical trials listed here were started after the abovementioned two laws were enacted in 2014, and some similar clinical studies were conducted before then under national ethics guidelines.

Clinical Trials That Transitioned from Preclinical Projects
As for iPS cells and ES cells, 10 out of 13 of the projects have started clinical trials. Although projects with iPS and ES cells are a little behind compared to somatic stem cells, the first-in-human tests are progressing smoothly. The most advanced is the retinal regeneration trial, and the results of two cases with autologous iPS cells (6 -1 ) and results with five cases using allogeneic iPS cells (6 -2 ) have already been published [45,46]. The original project was designed with autologous iPS cells, but due to the start of the iPS stock project, the clinical trial shifted toward the use of allogeneic iPS cells. Therefore, the trial with the autologous iPS cells was finished after the completion of the two cases, while the target sample size was five.   In the original protocol, the sample size was set at five, but because of the policy shift toward the use of allogeneic iPS cells, the project was finished when two cases were completed. For more details, see Supplementary Table S1 where the URL of each project is listed.
Another noteworthy clinical trial is the autologous cardiocyte transplantation for neonates with fatal congenital heart disease by Hidemasa Oh at Okayama University Hospital [53]. His strategy is to return patient's own cells isolated from tissue that was removed during neonatal cardioplasty to the cardiac tissue. The clinical trials are registered at ClinicalTrials.gov. as the TICAP prospective phase 1, PERSEUS randomized phase 2, APOLLON phase 3 randomized multicenter clinical trial, and the TICAP-DCM study.

Approved Stem Cell Therapies
Following the enforcement of the revised Act on Securing Quality, Efficacy and Safety of Products Including Pharmaceuticals and Medical Devices, 13 cell-processed products have been approved (Table 3). All products are based on the use of somatic cells or somatic stem cells, not on the use of iPS and ES cells yet. Japan Tissue Engineering is a pioneer company in the development of cell therapy in Japan and has two approved products, JACE and JACCthat have been developed as an autologous artificial skin for severe heat burn [54] and cartilage defects [55], respectively. Those two products were first approved in 2007 and 2012, respectively as a medical device in 2012, when the former unamended act had no category for the approval of regenerative medical products. After the reexamination for the expansion of indications, both were designated as human somatic stem cell-processed products. Table 3. List of regenerative medical products that have been approved by the Japanese government under the Act on Securing Quality, Efficacy and Safety of Products Including Pharmaceuticals and Medical Devices (compiled by the author based on the website of the Pharmaceuticals and Medical Devices Agency [56]).

No. Approval Date Nonproprietary Name, Material, and Indication Brand Name (Company) Decision
Human somatic cell-processed products   Chimeric antigen receptor-T (CAR-T) cell products and TEMCELL (cellular drug for acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation) were developed abroad and introduced.
After the revision of the act, TEMCELL and HeartSheet were the first items approved as regenerative medical products in 2015. HeartSheet is a cell sheet of autologous skeletal myoblast cultured on thermoresponsive hydrogels [57]. The sheets are applied on the heart surface of the patient with ischemic heart failure to induce revascularization in necrotic myocardial tissue. Nature dealt with the conditional approval of HeartSheet in an editorial [58]. Approval was given with seven cases, and it is expected that the number of cases will be increased to 60. The editorial posed whether it is acceptable to impose medical expenses to patients at this stage. It is hoped that the validity will be determined soon together with iPS cell-derived cardiomyocyte sheets.

Next Generation Research in Stem Cell Therapy
Preclinical research is progressing steadily. The transfer of genes such as LOTUS or DREADDs have enhanced the ability to repair spinal cord injuries using human iPS cell-derived neural stem cells [59,60]. Along with the central nervous system, effective stem cell therapy for peripheral nerve regeneration has been reported using a bio threedimension printer and xeno-free medium [61]. Approaches using stem cell therapy for the kidney and lung are also advancing. Transplantation of pig kidneys into monkeys requires strong immunosuppression but fetal porcine kidney survived with milder clinically applicable immunosuppression [62]. The transplanted kidney was vascularized by the host blood vessels, and this may work as an embryonic organ complementation site. A kidney precursor tissue, ureteric bud, was generated from human iPS cells [63]. The human iPS cell-derived lung progenitors were successfully integrated into mice lungs [64]. In addition, human tracheal tissue was created in vitro by combining cartilage, mesenchyme, and smooth muscle differentiation [65,66]. A new aspect of stem cell therapy is its use in the treatment of brain tumors. Glioblastoma is characterized by diffuse infiltration into the normal brain. Human iPS cells expressing a suicide gene had higher tumor-trophic migratory capacity and antitumor effects, indicating the potentiality of iPS cell-based therapy for invasive glioblastoma [67]. Induced tissue-specific stem (iTS) cells and dental pulp cells are unique and promising cells for therapy. Similar to iPS cells, iTS cells are produced by transient overexpression of the reprogramming factors but they still possess differentiation directivity to original cells, due to epigenetic memory [68]. iTS cells do not show full pluripotency, but have excellent differentiation directionality. Neural crest-origin dental pulp cells are attracting attention as a stem cell source [69]. Those cells obtained from deciduous teeth and young wisdom teeth are considered to have fewer ethical problems and higher stemness. These studies will be applied for clinical trials in the next decade.

Conclusions
A national program to support the transition of stem cell therapy into clinical practice has been in operation since 2011 in Japan, and 15 out of the 18 initial projects have started clinical trials. Along with the research budget, social frameworks, such as legisla-tion, a research headquarters, and an iPS cell stock were established. There are currently 13 approved cell-processed products. Cell therapy will face judgement on not only its effectiveness but also economic efficiency. Over the past decade, the social infrastructure for the development to the commercialization of regenerative medical products was established together with global harmonization. It is hoped that better products will be created in the near future based on this platform.

Conflicts of Interest:
The author declares no conflict of interest.