Safety Of Regenerative Medicine Research Articles

Autologous concentrated bone marrow injection for precollapse osteonecrosis of the femoral head concurrent with contralateral total hip arthroplasty: protocol for a clinical trial

IntroductionThe femoral head contralateral to the collapsed femoral head requiring total hip arthroplasty (THA) often manifests in the precollapse stage of osteonecrosis of the femoral head (ONFH). It is not...

Current status of platelet-rich plasma therapy under the act on the safety of regenerative medicine in Japan.

In Japan, a legal framework has been established for the safe and effective application of regenerative medicine. After eight years of the Act on the Safety of Regenerative Medicine (RM Act), discussions have been underway in the Ministry of Health, Labor and Welfare of Japan to revise the law owing to numerous novel technologies and inappropriate case reports not anticipated when the law was enacted. Therefore, in this review article, we have reviewed the regenerative medicine provision plans and the contribution of platelet-rich plasma (PRP) therapy, a regenerative medicine technique widely used in Japan post RM Act implementation, to these plans. As of January 2022, 97.2% of the regenerative medicine provided under the RM Act had been for private practice, and most of them were Class Ⅲ regenerative medicine. Notably, PRP was the most used processed cell under the RM Act. PRP therapy accounted for approximately 66% of the regenerative medicine provision plans in clinical research or private practice and was the most provided regenerative medicine technology in Japan. PRP therapy was primarily used in dentistry to regenerate periodontal tissue (approximately 50%), followed by orthopedics, where it is used to treat osteoarthritis. We suggest that further discussion is essential to determine the factors that should be addressed by the RM act to evaluate the efficacy and safety of PRP therapy.

Japanese Laws and the Current Status of Regenerative Medicine in the Tohoku Region.

The aim of this study was to review Japanese laws regarding regenerative medicine and the current status of clinical application of regenerative medicine, to learn about the advantages and problems, and to thereby serve as a reference for measures necessary for the development of regenerative medicine. Regenerative medicine started in 1957 with the transplantation of hematopoietic stem cells, followed by the establishment of embryonic stem cells in 1981 and induced pluripotent stem cells in 2006, and continues to evolve progressively. At the same time, however, problems have emerged due to lax legal regulations, such as the use of treatments that lack scientific evidence. The Japanese government enacted two laws to regulate regenerative medicine: the Law to Ensure the Safety of Regenerative Medicine and the Amend the Pharmaceutical Affairs Law in 2013. These laws were enacted with the aim of providing safe regenerative medicine promptly and smoothly and developing many regenerative medicine products. In these laws, regenerative medicine is defined as medical treatment that restores lost functions of damaged organs and tissues with the help of cellular and tissue-based products. Nowadays, there are two major methods of regenerative medicine. One representative method involves the transplantation of devices that activates self-regenerative ability by introducing living cells into patients' body. The other method is the activation and differentiation of endogenous stem cells with cell growth and differentiation factors. The current status of regenerative medicine in the Tohoku region after the enactment of these laws is described in detail. This clarified the advantages and disadvantages associated with regenerative medicine as it is currently practiced in Japan. Development of regenerative medicine in dentistry will be advanced by learning about its clinical application in medicine.

Neuroprotective effects of genome-edited human iPS cell-derived neural stem/progenitor cells on traumatic brain injury.

Despite developing neurosurgical procedures, few treatment options have achieved functional recovery from traumatic brain injury (TBI). Neural stem/progenitor cells (NS/PCs) may produce a long-term effect on neurological recovery. Although induced pluripotent stem cells (iPSCs) can overcome ethical and practical issues of human embryonic or fetal-derived tissues in clinical applications, the tumorigenicity of iPSC-derived populations remains an obstacle to their safe use in regenerative medicine. We herein established a novel treatment strategy for TBI using iPSCs expressing the enzyme-prodrug gene yeast cytosine deaminase-uracil phosphoribosyl transferase (yCD-UPRT). NS/PCs derived from human iPSCs displayed stable and high transgene expression of yCD-UPRT following CRISPR/Cas9-mediated genome editing. In vivo bioluminescent imaging and histopathological analysis demonstrated that NS/PCs concentrated around the damaged cortex of the TBI mouse model. During the subacute phase, performances in both beam walking test and accelerating rotarod test were significantly improved in the treatment group transplanted with genome-edited iPSC-derived NS/PCs compared with the control group. The injury area visualized by extravasation of Evans blue was smaller in the treatment group compared with the control group, suggesting the prevention of secondary brain injury. During the chronic phase, cerebral atrophy and ventricle enlargement were significantly less evident in the treatment group. Furthermore, after 5-fluorocytosine (5-FC) administration, 5-fluorouracil converted from 5-FC selectively eliminated undifferentiated NS/PCs while preserving the adjacent neuronal structures. NS/PCs expressing yCD-UPRT can be applied for safe regenerative medicine without the concern for tumorigenesis.

Difficulties in ensuring review quality performed by committees under the Act on the Safety of Regenerative Medicine in Japan.

We outlined five studies regarding the quality of the review by committees based on the Act on the Safety of Regenerative Medicine. The findings raise serious concerns about the independence, integrity, and quality of reviews of therapeutic plans by these committees with inappropriately close relationships to medical institutions and companies.

The First-in-Human Clinical Trial of iPSC-Derived Platelets (iPLAT1): Autologous Transfusion to an Aplastic Anemia Patient with Alloimmune Platelet Transfusion Refractoriness

Introduction: Platelet transfusion have saved lives of patients with thrombocytopenia through preventing or treating bleeding complications. Currently, platelet products are provided from blood banks which collect blood from healthy donors. However, our ageing society bears the risk of supply in the future. Furthermore, although the rate is decreasing, alloimmune platelet transfusion refractoriness (allo-PTR) is still found in 5% of platelet transfusion patients. Gestation and previous platelet transfusion cause sensitization to produce alloantibodies mostly against class I human leukocyte antigens (HLA-I) and less frequently against human platelet antigens (HPA), resulting in allo-PTR. In these cases, platelets from compatible donors are transfused, but for patients with rare HLA or HPA, donors are difficult to find.As a possible solution, we proposed the application of platelets from induced pluripotent stem cells (iPSC-platelets), which we have succeeded in the ex vivo production at clinical scale. iPSC-platelets are produced from expandable megakaryocyte cell lines (imMKCLs) as master cells and using a “turbulent flow” bioreactor and various new drugs. imMKCLs are established from iPSCs during megakaryocytic differentiation by overexpression of c-MYC, BMI1 and BCL-XL under the doxycycline control promoter. Switching off these transgenes leads to the maturation of imMKCLs. Turbulent flow was found to be a crucial factor in efficient ex vivo production of healthy iPSC-platelets from imMKCLs. We also developed TA-316, a thrombopoietin mimetic compound, KP-547, an ADAM17 inhibitor that inhibits CD42b shedding and stores platelet function, and also found the combination of AhR antagonist and ROCK inhibitor enables feeder-free liquid culture in imMKCL maturation.Aim: To evaluate the safety of autologous iPS-platelets administered to an aplastic anemia patient with allo-PTR due to anti-HPA alloantibody, who experienced systemic post-transfusion purpura-like complication and have no compatible donor in Japan.Methods and Results: Preclinical studies showed that iPSC-platelets were competent in in vitro assays and mouse and rabbit models for circulation and hemostasis, and pathogen and tumorigenicity free. The clinical study was approved by the Certified Special Committee for Regenerative Medicine of the Kyoto University and by the Health Sciences Council of the Japan Ministry of Health, Labour and Welfare as meeting the Act on the Safety of Regenerative Medicine. The clinical study started in March 2019. Using imMKCL master cells derived from the patient, iPS-platelets were manufactured at the Facility for iPS Cell Therapy (FiT) in Center for iPS Cell Research and Application (CiRA), Kyoto University according to the GMP standard. Three doses of 1x10^10, 3x10^10 and 1x10^11 were administered in a dose escalation single-center open-label uncontrolled study at Kyoto University Hospital. The primary endpoint was safety as measured by frequency and extent of adverse events, which were evaluated by the Efficacy Safety Assessment Committee for each dose cohort. Three doses of the administration of autologous iPS-platelets have been completed. No significant adverse event was observed during the full observation period of one year after the last dose.Conclusion: The iPLAT1 study completed the administration of iPSC-platelets for the first time and confirmed the safety in an allo-PTR patient who would otherwise have no HPA-compatible platelet product. The insights gained from the current study should further contribute to development of allogeneic iPSC-platelet products that can be readily administered to wide range of patients. (Japan Registry of Clinical Trials number jRCTa050190117) [Display omitted] DisclosuresSugimoto: Astellas Pharma Inc.: Honoraria; Ebara Corporation: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Megakaryon Co: Consultancy, Honoraria; Novartis Pharma K.K.: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria. Kanda: Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; SymBio Pharmaceuticals, Ltd.: Membership on an entity's Board of Directors or advisory committees; Sanofi K.K.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Ono Pharma Inc.: Honoraria; Novartis Pharma K.K.: Honoraria; NextGeM Inc: Patents & Royalties; Megakaryon Co: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Eisai: Research Funding; DAIICHI SANKYO Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; CHUGAI PHARMACEUTICAL Co., Ltd.: Honoraria; Bristol-Myers Squibb Co: Honoraria; Astellas Pharma Inc.: Consultancy, Honoraria; Amgen Astellas BioPharma: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria, Membership on an entity's Board of Directors or advisory committees; TEIJIN PHARMA LIMITED.: Honoraria. Kondo: Asahi Kasei Pharmaceutical: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Chugai Pharma: Honoraria; MSD Pharmaceutical: Honoraria; Sumitomo Dainippon Pharma: Honoraria. Shimizu: Megakaryon co: Consultancy. Hirai: Kyowa Kirin: Patents & Royalties, Research Funding; Bristol-Myers K.K.: Research Funding; CSL Behring: Research Funding; Mitsubishi Tanabe Pharma: Research Funding; Sumitomo Dainippon Pharma: Research Funding; Novartis Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding. Takaori-Kondo: Bristol-Myers K.K.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Research Funding; Celgene: Research Funding. Eto: Megakaryon co: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Kyowa Kirin Co., Ltd.: Honoraria; Takeda Pharmaceutical Co Ltd: Honoraria; Kowa Co Ltd: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; TEIJIN PHARMA LIMITED.: Honoraria; Kyoto Manufacturing Co., Ltd.: Research Funding.

Reflection on the enactment and impact of safety laws for regenerative medicine in Japan.

Japan's Act on the Safety of Regenerative Medicine (ASRM) created an innovative regulatory framework intended to safely promote the clinical development of stem cell-based interventions (SCBIs) while subjecting commercialized unproven SCBIs to greater scrutiny and accountability. This article reviews ASRM’s origins, explains its unprecedented scope, and assesses how it envisions the regulation of SCBIs. This analysis is used to highlight three key insights that are pertinent to the current revision of the ASRM: clarifying how the concept of safety should be defined and assessed in research and clinical care settings; revisiting risk criteria for review of SCBIs; and taking stronger measures to support the transition from unproven interventions to evidence-based therapies. Finally, the article reflects on lessons drawn from Japanese experiences in dealing with unproven SCBIs for international endeavors to regulate SCBIs.

Vinblastine treatment decreases the undifferentiated cell contamination of human iPSC-derived intestinal epithelial-like cells

Human induced pluripotent stem cell-derived intestinal epithelial cells (hiPSC-IECs) are expected to be utilized in regenerative medicine. To perform a safe transplantation without the risk of tumor formation, residual undifferentiated hiPSCs must be removed from hiPSC-IECs. In this study, we examined whether vinblastine (a multiple drug resistance 1 [MDR1] substrate) could remove residual undifferentiated hiPSCs in hiPSC-IECs and attempted to generate hiPSC-IECs applicable to transplantation medicine. We found that the expression levels of pluripotent markers were largely decreased and those of intestinal markers were increased by vinblastine treatment. The treatment of undifferentiated hiPSCs with vinblastine significantly decreased their viability. These results suggested that undifferentiated hiPSCs can be eliminated from hiPSC-IECs by vinblastine treatment. We hypothesized that MDR1-negative cells (such as undifferentiated hiPSCs) die upon vinblastine treatment because they are unable to excrete vinblastine. As expected, the cell viability of MDR1-knockout hiPSC-IECs was significantly decreased by vinblastine treatment. Furthermore, teratomas were formed by subcutaneous transplantation of hiPSC-IECs mixed with undifferentiated hiPSCs into mice, but they were not observed when the transplanted cells were pre-treated with vinblastine. Vinblastine-treated hiPSC-IECs would be an effective cell source for safe regenerative medicine.

Alterations of Glycosphingolipid Glycans and Chondrogenic Markers during Differentiation of Human Induced Pluripotent Stem Cells into Chondrocytes.

Due to the limited intrinsic healing potential of cartilage, injury to this tissue may lead to osteoarthritis. Human induced pluripotent stem cells (iPSCs), which can be differentiated into chondrocytes, are a promising source of cells for cartilage regenerative therapy. Currently, however, the methods for evaluating chondrogenic differentiation of iPSCs are very limited; the main techniques are based on the detection of chondrogenic genes and histological analysis of the extracellular matrix. The cell surface is coated with glycocalyx, a layer of glycoconjugates including glycosphingolipids (GSLs) and glycoproteins. The glycans in glycoconjugates play important roles in biological events, and their expression and structure vary widely depending on cell types and conditions. In this study, we performed a quantitative GSL-glycan analysis of human iPSCs, iPSC-derived mesenchymal stem cell like cells (iPS-MSC like cells), iPS-MSC-derived chondrocytes (iPS-MSC-CDs), bone marrow-derived mesenchymal stem cells (BMSCs), and BMSC-derived chondrocytes (BMSC-CDs) using glycoblotting technology. We found that GSL-glycan profiles differed among cell types, and that the GSL-glycome underwent a characteristic alteration during the process of chondrogenic differentiation. Furthermore, we analyzed the GSL-glycome of normal human cartilage and found that it was quite similar to that of iPS-MSC-CDs. This is the first study to evaluate GSL-glycan structures on human iPS-derived cartilaginous particles under micromass culture conditions and those of normal human cartilage. Our results indicate that GSL-glycome analysis is useful for evaluating target cell differentiation and can thus support safe regenerative medicine.

The Nature of Managing Regulations Regarding Regenerative Medicine in Japan

Background & Aim As the dramatic advancement of stem cell therapies ensures the value and trust of regenerative medicine in society, this requires increasingly robust ethicolegal considerations on these cell therapies. Like other counties, Japan has significantly addressed these considerations. As a result, Japan has led to establishing specific laws of regenerative medicine, where the Act on the Safety of Regenerative Medicine (ASRM) plays a key role in governing both clinical research and treatment of stem cell therapies. Since the ASRM is currently under revision, various scholars and policy makers are proactively approaching this process. On the other hand, the observation and analysis of the ASRM from a long-range perspective can provide new useful insights for its future nature and direction. Methods, Results & Conclusion From this perspective, in this study, we explore outstanding ethical and legal properties of the current Japanese regulations in question. As an example, our study sheds light on the approach of risk classifications using stem cells in the ASRM. While risk classifications of the ASRM, such as class 1 (high risk), class 2 (middle risk) and class 3 (low risk) emphasize technological aspects regarding stem cells, there is a possibility for this technological approach not necessarily to be effective or efficient in light of their review processes, such as safety assessments and operation processes. In our presentation, we will show more details of risk classifications in the ASRM, and furthermore, our research can be beneficial for the further promotion of stem cell therapies, along with a balance of participant’ rights, research and social value.

Regenerative Medicine and Drug Development using Stem Cells

There is an increasing interest in the induced pluripotent stem cells (iPSCs)-based regenerative medicine for various diseases. We have been developing regenerative medicine of spinal cord injury (SCI) by the transplantation of neural stem/progenitor cells (NS/PCs)-transplantation for many years. In a series of our previous efforts, we have addressed the issues of safety and tumorigenesis using iPSCs-derived NS/PCs (iPSCs-NS/PCs). The first-in-human clinical study of iPSC-based cell therapy for subacute SCI was approved by the government on February 18, 2019 as class I regenerative medicine protocol, which will be provided for under Japan's Act on the Safety of Regenerative Medicine.

Evaluation of Residual Human-Induced Pluripotent Stem Cells in Human Chondrocytes by Cell Type-Specific Glycosphingolipid Glycome Analysis Based on the Aminolysis-SALSA Technique.

Cartilage damage may eventually lead to osteoarthritis because it is difficult to repair. Human-induced pluripotent stem cell (iPSC)-derived chondrocytes may potentially be used to treat cartilage damage, but the tumorigenicity of iPSCs is a major concern for their application in regenerative medicine. Many glycoconjugates serve as stem cell markers, and glycosphingolipids (GSLs) including H type 1 antigen (Fucα1-2Galβ1-3GlcNAc) have been expressed on the surface of iPSCs. The purpose of the present study was to investigate whether GSL-glycome analysis is useful for quality control of residual iPSCs in chondrocytes. We performed GSL-glycome analysis of undifferentiated iPSCs in chondrocytes by combining glycoblotting and aminolysis-sialic acid linkage-specific alkylamidation (SALSA) method, enabling the detection of small quantities of iPSC-specific GSL-glycans from 5 × 104 cells. Furthermore, we estimated the residual amount of iPSCs using R-17F antibody, which possesses cytotoxic activity toward iPSCs that is dependent on the Lacto-N-fucopentaose I (LNFP I) of GSL. Moreover, we could detect a small number of LNFP I during mesenchymal stem cells (MSCs) differentiation from iPSCs. This is the first demonstration that GSL-glycome analysis is useful for detecting undifferentiated iPSCs, and can thereby support safe regenerative medicine.

Variation in the manufacturing reproducibility of autologous cell-based products depending on raw material shipment conditions.

To prepare an autologous cell-based product in a cell processing facility, the raw material, which is collected from a patient, must first be shipped from a medical institution to the facility. The quality of this raw material varies depending on the patient, and variations due to transport methods also occur. Because the quality must be uniform and manufacturing processes need to be adjusted to account for these variations, determining the effect of shipment conditions on raw materials is very important for estimating cell manufacturability in the process design. In this study, a group of medical institutions located in different areas requested similar cell-based products processed by the same manufacturing method to a company that is licensed under the Act on the Safety of Regenerative Medicine in Japan. Manufacturing reproducibility was analyzed based on 456 cell batches received from two clinics that were processed used the same manufacturing method. The specific growth rates that were observed in the early growth phase supposed that the proliferative potential of the primary cells in the raw material was influenced by transit time. Simultaneously, the variation of the specific growth rates in the late phase were supposed to be hardly occurred. Thus, this study evaluated shipping conditions of the raw materials for an autologous cell-based product, and a strategy for verifying the influence of transportation on quality in manufacturing was suggested.

Concise Review: Laying the Groundwork for a First-In-Human Study of an Induced Pluripotent Stem Cell-Based Intervention for Spinal Cord Injury.

There have been numerous attempts to develop stem cell transplantation approaches to promote the regeneration of spinal cord injury (SCI). Our multicenter team is currently planning to launch a first‐in‐human clinical study of an induced pluripotent stem cell (iPSC)‐based cell transplant intervention for subacute SCI. This trial was conducted as class I regenerative medicine protocol as provided for under Japan's Act on the Safety of Regenerative Medicine, using neural stem/progenitor cells derived from a clinical‐grade, integration‐free human “iPSC stock” generated by the Kyoto University Center for iPS Cell Research and Application. In the present article, we describe how we are preparing to initiate this clinical study, including addressing the issues of safety and tumorigenesis as well as practical problems that must be overcome to enable the development of therapeutic interventions for patients with chronic SCI. stem cells 2019;37:6–13

Current state and issues related to cell therapy under the Act on the Safety of Regenerative Medicine

In May the 2013, Regenerative Medicine Promotion Act was enacted in order to promote regenerative medicine in Japan. Based on this Act, in November 2014, the Act on the Safety of Regenerative Medicine, which ensured the safety of regenerative medicine, was put into effect. The Act on the Safety of Regenerative Medicine was intended to promote the swift and safe provision and promulgation of regenerative medicine, and it applies to both clinical research and private practice that utilizes regenerative medicine. In this review, an overview of the Act on the Safety of Regenerative Medicine is introduced, and the current state of this act and future issues are discussed.

The Change of Research and Development Environment by Act on the Safety of Regenerative Medicine and Revised Pharmaceutical Affairs Act:

The Change of Research and Development Environment by Act on the Safety of Regenerative Medicine and Revised Pharmaceutical Affairs Act:

[Current status of the regulation and development of cell therapy products in Japan].

Ten years have passed since Yamanaka et al. reported the establishment of human iPS cells, which became one of the triggers to make national efforts in Japan to promote research and translation of regenerative medicine and cell therapy (regenerative medicine etc.). However, it has been unreasonable in many cases to directly apply the existing regulation to cells processed for the purpose of use in regenerative medicine etc., which have quite different properties from conventional pharmaceuticals and medical devices. For this reason, in recent years, drastic reforms of various regulations of medical and pharmaceutical affairs have been vigorously pursued for efficient translation of regenerative medicine etc. Regarding medical affairs, "The Act on the Safety of Regenerative Medicine" was established for the purpose of providing safe regenerative medicine etc. to patients promptly and smoothly, establishing standards for regenerative medicine providing agencies and cell culture processing facilities. Regarding pharmaceutical affairs, a new chapter and an early approval system (conditional/term-limited approval system) for "regenerative medical products", which consists of cellular and gene therapy products, were introduced into "Pharmaceuticals and Medical Devices Act", a revised and renamed version of "Pharmaceutical Affairs Law". In this review article, we overview the current perspectives of regulations and challenges for translation of regenerative medicine etc. in Japan.

Ministerial ordinance revision of the Act on the Safety of Regenerative Medicine

Ministerial ordinance revision of the Act on the Safety of Regenerative Medicine

Comprehensive Quality Control of the Regenerative Therapy Using Platelet Concentrates: The Current Situation and Prospects in Japan.

Platelet concentrates (PCs), represented by platelet-rich plasma (PRP), have been widely applied in the fields of regenerative and aesthetic therapies. PCs' mechanisms of action, however, are too complicated, and it is not easy to present the whole picture; besides, clinical outcomes are hardly reproducible in many cases. Therefore, several medically advanced countries seemingly intend to regulate PC therapies weakly or strictly because of the increasing popularity. Japan established laws and regulations for PC therapy in the “Act on the Safety of Regenerative Medicine” along with the “Pharmaceuticals, Medical Devices and Other Therapeutic Products Act” in 2014, which, to our knowledge, represent the strictest regulatory framework for production and therapeutic use of PCs in the world. According to these laws and regulations, PCs produced for topical use should be prepared as cell-based medicinal products, essentially as should stem cells, in accordance with their registered (“licensed” under actual conditions) standard operating procedures. Nonetheless, criteria for their quality are not standardized. In this review, we discuss the quality of PC preparations by focusing on the basic concept and regulatory framework of regenerative medicine in Japan. Within the new framework, PC therapy is regulated by a specific notification and registration system, as is stem cell therapy. In comparison with the latter, however, risk factors that hamper successful PC therapy are much fewer. Via appropriate evaluation of patients' conditions and whole-blood samples by simple and sensitive but not yet fully standardized assays, it is theoretically possible that PC quality will be controlled nearly completely. In addition to or instead of standardization of preparation protocols, standardization of preoperative examination of individual PC preparations is an urgent task for improving and guaranteeing the safety and efficacy of PC therapy.

Morphological Analysis of Live Undifferentiated Cells Derived from Induced Pluripotent Stem Cells.

Induced pluripotent stem (iPS) cells possess pluripotency and self-renewal ability. Therefore, iPS cells are expected to be useful in regenerative medicine. However, iPS cells form malignant immature teratomas after transplantation into animals, even after differentiation induction. It has been suggested that undifferentiated cells expressing Nanog that remain after differentiation induction are responsible for teratoma formation. Various methods of removing these undifferentiated cells have therefore been investigated, but few methods involve morphological approaches, which may induce less cell damage. In addition, for cells derived from iPS cells to be applied in regenerative medicine, they must be alive. However, detailed morphological analysis of live undifferentiated cells has not been performed. For the above reasons, we assessed the morphological features of live undifferentiated cells remaining after differentiation induction as a basic investigation into the clinical application of iPS cells. As a result, live undifferentiated cells remaining after differentiation induction exhibited a round or oval cytoplasm about 12 μm in diameter and a nucleus. They exhibited nucleo-cytoplasmic (N/C) ratio of about 60% and eccentric nuclei, and they possessed partially granule-like structures in the cytoplasm and prominent nucleoli. Although they were similar to iPS cells, they were smaller than live iPS cells. Furthermore, very small cells were present among undifferentiated cells after differentiation induction. These results suggest that the removal of undifferentiated cells may be possible using the morphological features of live iPS cells and undifferentiated cells after differentiation induction. In addition, this study supports safe regenerative medicine using iPS cells.