H1FOO-DD promotes efficiency and uniformity in reprogramming to naive pluripotency.

Heterogeneity among both primed and naive pluripotent stem cell lines remains a major unresolved problem. Here we show that expressing the maternal-specific linker histone H1FOO fused to a destabilizing domain (H1FOO-DD), together with OCT4, SOX2, KLF4, and LMYC, in human somatic cells improves the quality of reprogramming to both primed and naive pluripotency. H1FOO-DD expression was associated with altered chromatin accessibility around pluripotency genes and with suppression of the innate immune response. Notably, H1FOO-DD generates naive induced pluripotent stem cells with lower variation in transcriptome and methylome among clones and a more uniform and superior differentiation potency. Furthermore, we elucidated that upregulation of FKBP1A, driven by these five factors, plays a key role in H1FOO-DD-mediated reprogramming.

A clinical-grade HLA haplobank of human induced pluripotent stem cells matching approximately 40% of the Japanese population.

BACKGROUND: Human induced pluripotent stem cells (iPSCs) are expected to be useful for regenerative medicine for many diseases. Many researchers have focused on and enabled the generation of differentiated cells or tissue-like structures, including organoids, which help to ameliorate target diseases. To promote such cell therapies, we established a clinically applicable iPSC haplobank matching as many people as possible in Japan. METHODS: Through cooperation with several organizations, we recruited donors whose human leukocyte antigens (HLAs) involved in immunorejection were homozygous. The peripheral or umbilical cord blood collected from the donors was used for iPSC production by electroporation of episomal vectors. These iPSC lines were then subjected to testing, including genome analyses and sterility, to maximize safety. FINDINGS: We constructed a clinical-grade haplobank of 27 iPSC lines from 7 donors according to good manufacturing practice regulations. However, reasons to avoid using iPSC lines include the presence of residual episomal vectors or genetic mutations in cancer-related genes. CONCLUSIONS: This haplobank provides HLA-matched iPSC lines for approximately 40% of the Japanese population. Since the haplobank's release in 2015, these iPSC lines have been used in more than 10 clinical trials. The establishment of this haplobank is an important step toward the clinical application of iPSCs in cell therapies. FUNDING: This study was supported by a research center network for the realization of regenerative medicine of the Japan Agency for Medical Research and Development (AMED) under grant number JP20bm0104001h0108.

Improved Sendai viral system for reprogramming to naive pluripotency.

Naive human induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells with Sendai virus (SeV) vectors. However, only dermal fibroblasts have been successfully reprogrammed this way, and the process requires culture on feeder cells. Moreover, SeV vectors are highly persistent and inhibit subsequent differentiation of iPSCs. Here, we report a modified SeV vector system to generate transgene-free naive human iPSCs with superior differentiation potential. The modified method can be applied not only to fibroblasts but also to other somatic cell types. SeV vectors disappear quickly at early passages, and this approach enables the generation of naive iPSCs in a feeder-free culture. The naive iPSCs generated by this method show better differentiation to trilineage and extra-embryonic trophectoderm than those derived by conventional methods. This method can expand the application of iPSCs to research on early human development and regenerative medicine.

Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application.

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.

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.

Transplantation of human autologous synovial mesenchymal stem cells with trisomy 7 into the knee joint and 5 years of follow-up.

Mesenchymal stem cells (MSCs) can show trisomy 7; however, the safety of these cells has not been fully investigated. The purposes of this study were to determine the ratio of patients whose synovial MSCs were transplanted clinically, to intensively investigate MSCs with trisomy 7 from a safety perspective, and to follow up the patients for 5 years after transplantation. Synovial MSCs at passage 0 were transplanted into a knee for degenerative meniscus tears in 10 patients, and the patients were checked at 5 years. The synovial MSCs were evaluated at passages 0 to 15 by G-bands and digital karyotyping, and trisomy 7 was found in 3 of 10 patients. In those three patients, 5% to 10% of the synovial MSCs showed trisomy 7. The mRNA expressions of representative oncogenes and genes on chromosome 7 did not differ between MSCs with and without trisomy 7. Whole-genome sequencing and DNA methylation analysis showed similar results for MSCs with and without trisomy 7. Transplantation of human synovial MSCs with trisomy 7 into eight mouse knees did not result in tumor formation under the skin or in the knees after 8 weeks in any mouse, whereas transplanted HT1080 cells formed tumors. In vitro chondrogenic potentials were similar between MSCs with and without trisomy 7. Five-year follow-ups revealed no serious adverse events in all 10 human patients, including 3 who had received MSCs with trisomy 7. Overall, our findings indicated that synovial MSCs with trisomy 7 were comparable with MSCs without trisomy 7 from a safety perspective.

Non-clinical efficacy, safety and stable clinical cell processing of induced pluripotent stem cell-derived anti-glypican-3 chimeric antigen receptor-expressing natural killer/innate lymphoid cells.

The use of allogeneic, pluripotent stem-cell-derived immune cells for cancer immunotherapy has been the subject of recent clinical trials. In Japan, investigator-initiated clinical trials will soon begin for ovarian cancer treatment using human leukocyte antigen (HLA)-homozygous-induced pluripotent stem cell (iPSC)-derived anti-glypican-3 (GPC3) chimeric antigen receptor (CAR)-expressing natural killer/innate lymphoid cells (NK/ILC). Using pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, in terms of efficacy, safety and producibility. In this paper, we describe our methods for the stable, feeder-free production of CAR-expressing NK/ILC cells from CAR-transduced iPSC with clinically relevant scale and materials. The average number of cells that could be differentiated from 1.8-3.6 × 106 iPSC within 7 weeks was 1.8-4.0 × 109 . These cells showed stable CD45/CD7/CAR expression, effector functions of cytotoxicity and interferon gamma (IFN-γ) production against GPC3-expressing tumor cells. When the CAR-NK/ILC cells were injected into a GPC3-positive, ovarian-tumor-bearing, immunodeficient mouse model, we observed a significant therapeutic effect that prolonged the survival of the animals. When the cells were injected into immunodeficient mice during non-clinical safety tests, no acute systemic toxicity or tumorigenicity of the final product or residual iPSC was observed. In addition, our test results for the CAR-NK/ILC cells generated with clinical manufacturing standards are encouraging, and these methods should accelerate the development of allogeneic pluripotent stem cell-based immune cell cancer therapies.

CiRA iPSC seed stocks (CiRA's iPSC Stock Project).

In Japan, induced pluripotent stem cell (iPSC) Stock Project has started from 2013. The goal of the Project is to manufacture and release clinical-grade HLA homozygous iPSC lines that can cover almost the entire of Japanese population. We show the summary of the cell lines distributed, test results for quality control and future plans.

Overview: an iPS cell stock at CiRA.

Induced pluripotent stem cells (iPSCs) can be produced from various somatic cells and have the ability to differentiate into various cells and tissues of the body. Regenerative medicine using iPSCs is expected to manage diseases lacking effective treatments at present. We are establishing a safe and effective iPSC stock that can be used for regenerative medicine. Our iPSC stock is recruited from healthy, consenting HLA-type homozygous donors and is made with peripheral blood-derived mononuclear cells or umbilical cord blood. We hope to minimize the influence of immune rejection by preparing HLA homozygous iPSCs. Our stock is made at the Cell Processing Center (CPC), Center for iPS Cell Research and Application (CiRA). We are preparing iPS cells that maximize matching of the Japanese population at the major HLA loci. This iPSC stock is intended to be offered not only to Japanese centers but also overseas medical institutions and companies. In August 2015, we began offering the iPSC stock for regenerative medicine and now offer 21 clones made from 5 donors.

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 .).

[Present and Future of an iPS Cell Stock for Regenerative Medicine].

A haplobank of induced pluripotent stem cell (iPSC) lines derived from healthy donors with homozygous human leukocyte antigen(HLA), which is called "an iPSC stock", has been under construction in Japan. The iPSC stock is expected to enable HLA matching of a majority of recipients and reduce the risk of trans- plant rejection. Full-scale operations began in FY2013, with the aim of covering 30-50% of the Japanese population by FY2017 and most of the population by FY2022 with the iPSC stock. A novel feeder-free and xeno-free culture system for iPSCs was developed to comply with regulatory safety standards. In 2015, a clinical-grade iPSC line with homozygous HLA of the highest-frequency haplotype was released as a first in the world. Other clinical-grade lines are being generated successively. If all goes to plan, the first clinical research using the iPSC stock will start in 2017. However, many challenges remain to ensuring the future of iPSC stock. In accordance with the progress of the latest research, safety issues regarding iPSC-based cell therapy are being monitored with much interest, and one of the major concerns is tumorigenicity. We have to continue to discuss the extent of genomic abnormalities that would or would not be acceptable in not only iPSC-derived products but also iPSC stock, taking into account the risks and benefits of cell therapy. It is also necessary to demonstrate the clinical efficacy of HLA matching, which is indicated to promote graft survival and reduce immunosuppressive drug use.