Considerations for the development of iPSC-derived cell therapies: a review of key challenges by the JSRM-ISCT iPSC Committee.

Since their first production in 2007, human induced pluripotent stem cells (iPSCs) have provided a novel platform for the development of various cell therapies targeting a spectrum of diseases, ranging from rare genetic eye disorders to cancer treatment. However, several challenges must be tackled for iPSC-based cell therapy to enter the market and achieve broader global adoption. This white paper, authored by the Japanese Society for Regenerative Medicine (JSRM) - International Society for Cell Therapy (ISCT) iPSC Committee delves into the hurdles encountered in the pursuit of safe and economically viable iPSC-based therapies, particularly from the standpoint of the cell therapy industry. It discusses differences in global guidelines and regulatory frameworks, outlines a series of quality control tests required to ensure the safety of the cell therapy, and provides details and important considerations around cost of goods (COGs), including the impact of automated advanced manufacturing.

Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration.

We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number, UMIN000011929 .).

A Report from a Workshop of the International Stem Cell Banking Initiative, Held in Collaboration of Global Alliance for iPSC Therapies and the Harvard Stem Cell Institute, Boston, 2017.

This report summarizes the recent activity of the International Stem Cell Banking Initiative held at Harvard Stem Cell Institute, Boston, MA, USA, on June 18, 2017. In this meeting, we aimed to find consensus on ongoing issues of quality control (QC), safety, and efficacy of human pluripotent stem cell banks and their derivative cell therapy products for the global harmonization. In particular, assays for the QC testing such as pluripotency assays test and general QC testing criteria were intensively discussed. Moreover, the recent activities of global stem cell banking centers and the regulatory bodies were briefly summarized to provide an overview on global developments and issues. Stem Cells 2019;37:1130-1135.

Report of the international conference on manufacturing and testing of pluripotent stem cells.

Sessions included an overview of past cell therapy (CT) conferences sponsored by the International Alliance for Biological Standardization (IABS). The sessions highlighted challenges in the field of human pluripotent stem cells (hPSCs) and also addressed specific points on manufacturing, bioanalytics and comparability, tumorigenicity testing, storage, and shipping. Panel discussions complemented the presentations. The conference concluded that a range of new standardization groups is emerging that could help the field, but ways must be found to ensure that these efforts are coordinated. In addition, there are opportunities for regulatory convergence starting with a gap analysis of existing guidelines to determine what might be missing and what issues might be creating divergence. More specific global regulatory guidance, preferably from WHO, would be welcome. IABS and the California Institute for Regenerative Medicine (CIRM) will explore with stakeholders the development of a practical and innovative road map to support early CT product (CTP) developers.

Protective Effects of Human iPS-Derived Retinal Pigmented Epithelial Cells in Comparison with Human Mesenchymal Stromal Cells and Human Neural Stem Cells on the Degenerating Retina in rd1 mice.

Retinitis pigmentosa (RP) is a group of visual impairments characterized by progressive rod photoreceptor cell loss due to a genetic background. Pigment epithelium-derived factor (PEDF) predominantly secreted by the retinal pigmented epithelium (RPE) has been reported to protect photoreceptors in retinal degeneration models, including rd1. In addition, clinical trials are currently underway outside Japan using human mesenchymal stromal cells and human neural stem cells to protect photoreceptors in RP and dry age-related macular degeneration, respectively. Thus, this study aimed to investigate the rescue effects of induced pluripotent stem (iPS)-RPE cells in comparison with those types of cells used in clinical trials on photoreceptor degeneration in rd1 mice. Cells were injected into the subretinal space of immune-suppressed 2-week-old rd1 mice. The results demonstrated that human iPS-RPE cells significantly attenuated photoreceptor degeneration on postoperative days (PODs) 14 and 21 and survived longer up to at least 12 weeks after operation than the other two types of graft cells with less immune responses and apoptosis. The mean PEDF concentration in the intraocular fluid in RPE-transplanted eyes was more than 1 µg/ml at PODs 14 and 21, and this may have contributed to the protective effect of RPE transplantation. Our findings suggest that iPS-RPE cells serve as a competent source to delay photoreceptor degeneration through stable survival in degenerating ocular environment and by releasing neuroprotective factors such as PEDF.

Report of the International Regulatory Forum on Human Cell Therapy and Gene Therapy Products.

The development of human cell therapy and gene therapy products has progressed internationally. Efforts have been made to address regulatory challenges in the evaluation of quality, efficacy, and safety of the products. In this forum, updates on the specific challenges in quality, efficacy, and safety of products in the view of international development were shared through the exchange of information and opinions among experts from regulatory authorities, academic institutions, and industry practitioners. Sessions identified specific/critical points to consider for the evaluation of human cell therapy and gene therapy products that are different from conventional biological products; common approaches and practices among regulatory regions were also shared. Certain elements of current international guidelines might not be appropriate to be applied to these products. Further, international discussion on the concept of potency and in vivo tumorigenicity studies, among others, is needed. This forum concluded that the continued collective actions are expected to promote international convergence of regulatory approaches of the products. The Pharmaceuticals and Medical Devices Agency and Japanese Society for Regenerative Medicine jointly convened the forum with support from the National Institutes of Biomedical Innovation, Health and Nutrition. Participants at the forum include 300 experts in and outside of Japan.

Human Gingival Integration-Free iPSCs; a Source for MSC-Like Cells.

Mesenchymal stem cells (MSCs) are considered a potential autologous therapy for tissue engineering. The available procedures for MSC retrieval from patients are invasive, and their limited in vitro proliferation restricts their use in the treatment of damaged tissues. Therefore, it is important to establish an alternative and safe source of MSCs. The objective of this study was to demonstrate induced pluripotent stem cell (iPSC) generation from a combination of an accessible source tissue and an integration-free method; we also attempted the differentiation of iPSCs into MSC-like cells (MSLCs) for future autologous tissue engineering. iPSCs were derived from human gingival tissues, which are easily accessible in the field of dentistry, via the use of non-integrating episomal plasmids. Established iPSCs expressed embryonic stem (ES) cell-specific markers, as assessed by gene analysis and immunocytochemistry. Embryoid bodies and teratoma formation were formed from iPSCs, showing their capacity to differentiate into three germ layers. Furthermore, we were successful in differentiating iPSCs into MSLCs. They tested positively for their capacity of trilineage differentiation. Our results demonstrate that human gingival integration-free iPSCs, readily accessible stem cells generated using episomal plasmid vectors, are a promising source of MSLCs, which can be used in tissue regeneration.

Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors.

After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34(+) cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgene-free iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future.

Study on the Extrapolability of Current Tumorgenicity Test With Mice by Comparing the Syngeneic or Allogeneic Mouse Transplantation Model.

The extrapolability of the current tumorigenicity test performed by transplanting human cell product into immunodeficient (NOG) mice was investigated. For this purpose, the susceptibility to form teratomas of NOG mice was assessed by transplanting undifferentiated human-induced pluripotent stem cells (hiPSCs) as positive control cells via the liver, striatum, or tail vein and evaluating the TPD50 value (dose required to form teratomas in half of the transplanted mice). This was then compared to the TPD50 of syngeneic or allogeneic mouse models. The TPD50 of C57/BL/6(B6)-iPSC or 129/Ola(129)-embryonic stem cell (ESC) transplanted into the liver of syngeneic mice was 4.08 × 105 and 4.64 × 104 cells, respectively, while the TPD50 of hiPSC administered into the liver of NOG mice was 4.64 × 104 cells. The TPD50 of B6-miPSC-synergic, 129-mESC-synergic, or 129-cell/B6 allogeneic transplantation into the striatum was 5.09 × 102, 1.0 × 104, and 3.73 × 104 cells, respectively, while that of hiPSC/NOG mice was 1.0 × 103 cells. The TPD50 for B6-miPSC or 129-mESC syngeneic tail vein infusion was 3.16 × 106 or 5.62 × 106 cells, respectively, while no incidence was observed from 1 × 107 B6-miPSCs in 129 mice or hiPSCs in NOG mice infusion study. Although the number of data sets was limited, these data indicate that the teratoma formation from transplanted undifferentiated hiPSCs via the liver or striatum in NOG mice is comparable to that in syngeneic or allogeneic mouse transplantation model, suggesting that the result of the current tumorigenicity test in NOG mice would provide useful information to infer the incidence of teratoma from residual undifferentiated hPSCs in hPSC-derived products after transplantation.

Challenges in Global Access to CAR-T cells: an Asian Perspective.

The use of cell therapy for clinical applications has seen a dramatic increase in recent years, primarily in oncology, especially with the use of chimeric antigen receptor (CAR) T-cell therapies. However, there are some barriers to the widespread adoption of CAR-T cell therapies globally, primarily because of the high cost of manufacturing these cells and clinical infrastructure considerations. We reviewed the different strategies adopted across Asia to implement CAR-T cell therapy and found that these included patient assistance programs, close engagement with funders, cost-effectiveness studies, on-site manufacturing of CAR-T cells, and joint ventures between local partners and foreign pharmaceutical companies. Although on-site manufacturing can reduce the cost of genetic engineering and expansion, it does not address many other hidden costs and quality considerations. Future growth in large-scale regional manufacturing, facilitated by cutting-edge science and innovation, could reduce costs through economies of scale and facilitate the eagerly needed global access.

A Novel Approach for Determining the Critical Quality Attributes of Mesenchymal Stem Cells by Specifying Cell Population With Replication Potential.

We introduce a novel approach to determine the critical quality attributes (CQAs) of mesenchymal stem cells (MSCs) expected to exert immunosuppressive effects. MSCs retained homeostatic replication potentials, such as sustainable growth and consistent cell morphology as a population, in early passages, but lost them in late passages. Characteristic surface markers of MSCs (ie, CD73, CD90, and CD105) were no longer expressed at 2 weeks after subcutaneous transplantation into NOG mice when MSCs from late passages were transplanted, but not when MSCs from early passages were transplanted, suggesting that the biological effects of the MSCs differed according to the timing of cell harvesting and highlighting the importance of specifying MSCs that retained homeostatic features to define the CQAs. The homeostatic features of MSCs related to the balance of the redox system, nutrient requirements, and mitochondrial function were also observed until a certain passage. Therefore, we could define the CQAs of MSCs related to manufacturing by selecting process parameters (PPs) underlying the homeostatic features of MSCs and measuring these PPs quantitatively to specify the cell population with homeostatic features by limiting the passage number. The validity of the PPs stipulated in our pilot study was verified using an SKG murine arthritis model, and critical PPs (CPPs) were then selected among the PPs. Thus, CQAs related to manufacturing in the developmental phase could be defined by the CPPs in this manner, and the concept of CQAs could be refined continuously toward commercial manufacturing.

Self-organization, quality control, and preclinical studies of human iPSC-derived retinal sheets for tissue-transplantation therapy.

Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human pluripotent stem cells (hPSCs). Here we present a quality control method and preclinical studies for tissue-sheet transplantation. Self-organizing hPSCs differentiated into both retinal and off-target tissues. Gene expression analyses identified the major off-target tissues as eye-related, cortex-like, and spinal cord-like tissues. For quality control, we developed a qPCR-based test in which each hPSC-derived neuroepithelium was dissected into two tissue-sheets: inner-central sheet for transplantation and outer-peripheral sheet for qPCR to ensure retinal tissue selection. During qPCR, tissue-sheets were stored for 3-4 days using a newly developed preservation method. In a rat tumorigenicity study, no transplant-related adverse events were observed. In retinal degeneration model rats, retinal transplants differentiated into mature photoreceptors and exhibited light responses in electrophysiology assays. These results demonstrate our rationale toward self-organizing retinal sheet transplantation therapy.

Improving the differentiation potential of pluripotent stem cells by optimizing culture conditions.

Embryoid cells and induced pluripotent stem cells (iPSCs) are pluripotent stem cells (PSCs). They retain differentiation and self-renewal potential. However, the differentiation potential of PSCs can be changed by the culture medium. PSCs retain their differentiation potential when cultured with medium that supports the glycolytic pathway, showing high expression of chromodomain-helicase-DNA-binding protein 7 (CHD7), but lose their differentiation potential with medium that supports mitochondrial function, showing reduced levels of CHD7. Labeling cells by their copy number variant profile revealed that genetically different PSC populations can be cultured by medium selection. Another factor that defines the self-renewal potential of PSCs is culture condition. PSCs form colonies as they grow, and spontaneous differentiation inevitably occurs along the rim of these colonies in areas that lack cell-to-cell contact; because of this, undifferentiated cell populations would diminish if differentiated cells are not removed properly. Seeding cells on a less potent cell-binding material may minimize the inclusion of differentiated cells, exploiting the reduced adhesive properties of differentiated cells. Culturing cells with medium that supports the glycolytic pathway, using CHD7 as a biomarker for differentiation potential, and culturing cells on less sticky material can improve the differentiation potential of already established PSC clones.

A phase I/IIa clinical trial of third-generation autologous chondrocyte implantation (IK-01) for focal cartilage injury of the knee.

Background/objective: The purpose of this study was to report the outcomes of a clinical trial conducted in Japan to assess the safety and effectiveness of third-generation autologous chondrocyte implantation (ACI) using IK-01 (CaReS™), which does not require flap coverage, in the treatment of patients with focal cartilage injury of the knee. Methods: This was an open label, exploratory clinical trial. Patients were enrolled between June 2012 and September 2016. The primary endpoint of the study was the International Knee Documentation Committee (IKDC) score at 52 weeks after implantation. The IKDC, Lysholm, and visual analog scale (VAS) scores were evaluated at the time of screening and at 4, 12, 24, 36, and 52 weeks after implantation. Improvements from the baseline scores were evaluated using the equation "(postoperative score) - (preoperative score)." Magnetic resonance imaging (MRI) was performed at 2, 12, 24, and 52 weeks after implantation, and MRI measurements were evaluated using T1 rho and T2 mapping. Results: Nine patients were enrolled in this study and were examined for safety. Product quality did not satisfy the specification in one patient, and bacterial joint infection occurred in one patient. As a result, seven patients were included in the outcome analyses. The mean IKDC score significantly improved from 36.4 preoperatively to 74.1% at 52 weeks after implantation (p < 0.0001). The mean Lysholm and VAS scores also significantly improved from 39.6 to 57.4 to 89.6 and 22.9, respectively, after surgery. In the MRI evaluation, the T1 rho and T2 values of the implanted area were similar to those of the surrounding cartilage at 52 weeks after implantation. Conclusions: Third generation ACI (IK-01) can be an effective treatment option for focal cartilage defects of the knee; however, surgeons must pay careful attention to the risk of postoperative joint infection.

Correlation Between Genetic Abnormalities in Induced Pluripotent Stem Cell-Derivatives and Abnormal Tissue Formation in Tumorigenicity Tests.

Cell therapy using induced pluripotent stem cell (iPSC) derivatives may result in abnormal tissue generation because the cells undergo numerous cycles of mitosis before clinical application, potentially increasing the accumulation of genetic abnormalities. Therefore, genetic tests may predict abnormal tissue formation after transplantation. Here, we administered iPSC derivatives with or without single-nucleotide variants (SNVs) and deletions in cancer-related genes with various genomic copy number variant (CNV) profiles into immunodeficient mice and examined the relationships between mutations and abnormal tissue formation after transplantation. No positive correlations were found between the presence of SNVs/deletions and the formation of abnormal tissues; the overall predictivity was 29%. However, a copy number higher than 3 was correlated, with an overall predictivity of 86%. Furthermore, we found CNV hotspots at 14q32.33 and 17q12 loci. Thus, CNV analysis may predict abnormal tissue formation after transplantation of iPSC derivatives and reduce the number of tumorigenicity tests.

Data on long-term survival of the NOD/Shi-scid IL-2Rγnull (NOG) mouse in two facilities.

The objective of this study was to investigate an appropriate observation period for an evaluation of tumorigenicity in NOD/Shi-scid IL-2 Rγnull (NOG) mice. At SNBL, 19 male and 19 female NOG mice were observed the general condition from 7 weeks old up to 68 weeks old and at FBRI, 7 male and 16 female NOG mice were observed the general condition throughout the lifespan from 7 weeks old. The survival rate started to decline rapidly around 54 to 56 weeks of age in both facilities without a facility difference. Based on these survival data, it seems reasonable to terminate a tumorigenicity study at 52 weeks of age.

First-in-human clinical trial of transplantation of iPSC-derived NS/PCs in subacute complete spinal cord injury: Study protocol.

INTRODUCTION: Our group has conducted extensive basic and preclinical studies of the use of human induced pluripotent cell (iPSC)-derived neural stem/progenitor cell (hiPSC-NS/PC) grafts in models of spinal cord injury (SCI). Evidence from animal experiments suggests this approach is safe and effective. We are preparing to initiate a first-in-human clinical study of hiPSC-NS/PC transplantation in subacute SCI. SETTING: NS/PCs were prepared at a Good Manufacturing Practice-grade cell processing facility at Osaka National Hospital using a clinical-grade integration-free hiPSC line established by the iPSC Stock Project organized by the Kyoto University Center for iPS Cell Research and Application. After performing all quality checks, the long-term safety and efficacy of cells were confirmed using immunodeficient mouse models. METHODS: The forthcoming clinical study uses an open-label, single-arm design. The initial follow-up period is 1 year. The primary objective is to assess the safety of hiPSC-NS/PC transplantation in patients with subacute SCI. The secondary objective is to obtain preliminary evidence of its impact on neurological function and quality-of-life outcomes. Four patients with C3/4-Th10 level, complete subacute (within 24 days post-injury) SCI will be recruited. After obtaining consent, cryopreserved cells will be thawed and prepared following a multi-step process including treatment with a γ-secretase inhibitor to promote cell differentiation. A total of 2 × 106 cells will be transplanted into the injured spinal cord parenchyma 14-28 days post-injury. Patients will also receive transient immunosuppression. This study protocol has been reviewed and approved by the Certified Committee for Regenerative Medicine and the Japanese Ministry of Health, Labor and Welfare (University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number, UMIN000035074; Japan Registry of Clinical Trials [jRCT] number, jRCTa031190228). DISCUSSION/CONCLUSION: We plan to start recruiting a patient as soon as the COVID-19 epidemic subsides. The primary focus of this clinical study is safety, and the number of transplanted cells may be too low to confirm efficacy. After confirming safety, a dose-escalation study is planned.

Human keratinocytes derived from the bulge region of hair follicles are refractory to differentiation.

Human keratinocyte strains derived from the bulge region of plucked human follicles were successfully established from all 43 donors (age 24-76) regardless of the age and gender. The total cell number, number of population doublings and population doubling time were similar among the strains. These bulge-derived keratinocytes, BDKs, expressed keratin family genes specific to basal cell layers of the epidermis. They also expressed CD34, one of the bulge stem cell marker genes. The growth behavior and positivity of CD34 indicate that BDKs contain stem cells. BDKs were cultured until confluency or treated with CaCl2 to induce differentiation. Morphology and expression of keratin family genes in BDKs before and after differentiation induction with CaCl2 were similar to those of epidermal keratinocytes obtained from skin biopsies (NHEKs). However, expression levels of keratin-10, a prickle cell layer marker, in CaCl2-treated BDKs were lower than those in CaCl2-treated NHEKs. Higher expression of integrin-alpha6, a basal cell layer marker, was also noted in BDKs than in NHEKs after differentiation induction. Expression of stem cell marker genes other than CD34, including CD200, Sox2 and NANOG, was about the same at confluency in both cells, but significantly higher in BDKs than NHEKs after differentiation. These results indicate that BDKs were more refractory to differentiation than NHEKs. We then examined Wnt signaling inhibitor genes, DKK-3 and WIF-1 that function as tumor suppressors. DKK-3 expression decreased in both BDKs and NHEKs after CaCl2-induced differentiation. Expression of WIF-1 decreased 50% in BDKs one day after CaCl2 treatment and remained low, but was induced 1.7 times in NHEKs one day after CaCl2 treatment and further induced thereafter (>2.5 times), suggesting that WIF-1 may be involved in maintaining the differentiation-refractory status of BDKs. Since cancer stem cells in the skin have been reported to be similar to bulge-derived stem cells, our BDK strains may be of use in studying characteristics of cancer stem cells of the epidermis.

Generation and Molecular Characterization of Human Ring Sideroblasts: a Key Role of Ferrous Iron in Terminal Erythroid Differentiation and Ring Sideroblast Formation.

Ring sideroblasts are a hallmark of sideroblastic anemia, although little is known about their characteristics. Here, we first generated mutant mice by disrupting the GATA-1 binding motif at the intron 1 enhancer of the ALAS2 gene, a gene responsible for X-linked sideroblastic anemia (XLSA). Although heterozygous female mice showed an anemic phenotype, ring sideroblasts were not observed in their bone marrow. We next established human induced pluripotent stem cell-derived proerythroblast clones harboring the same ALAS2 gene mutation. Through coculture with sodium ferrous citrate, mutant clones differentiated into mature erythroblasts and became ring sideroblasts with upregulation of metal transporters (MFRN1, ZIP8, and DMT1), suggesting a key role for ferrous iron in erythroid differentiation. Interestingly, holo-transferrin (holo-Tf) did not induce erythroid differentiation as well as ring sideroblast formation, and mutant cells underwent apoptosis. Despite massive iron granule content, ring sideroblasts were less apoptotic than holo-Tf-treated undifferentiated cells. Microarray analysis revealed upregulation of antiapoptotic genes in ring sideroblasts, a profile partly shared with erythroblasts from a patient with XLSA. These results suggest that ring sideroblasts exert a reaction to avoid cell death by activating antiapoptotic programs. Our model may become an important tool to clarify the pathophysiology of sideroblastic anemia.