Epigenetic-scale comparison of human iPSCs generated by retrovirus, Sendai virus or episomal vectors

Human induced pluripotent stem cells (iPSCs) are established by introducing several reprogramming factors, such as OCT3/4, SOX2, KLF4, c-MYC. Because of their pluripotency and immortality, iPSCs are considered to be a powerful tool for regenerative medicine. To date, iPSCs have been established all over the world by various gene delivery methods. All methods induced high-quality iPSCs, but epigenetic analysis of abnormalities derived from differences in the gene delivery methods has not yet been performed. Here, we generated genetically matched human iPSCs from menstrual blood cells by using three kinds of vectors, i.e., retrovirus, Sendai virus, and episomal vectors, and compared genome-wide DNA methylation profiles among them. Although comparison of aberrant methylation revealed that iPSCs generated by Sendai virus vector have lowest number of aberrant methylation sites among the three vectors, the iPSCs generated by non-integrating methods did not show vector-specific aberrant methylation. However, the differences between the iPSC lines were determined to be the number of random aberrant hypermethylated regions compared with embryonic stem cells. These random aberrant hypermethylations might be a cause of the differences in the properties of each of the iPSC lines

A safeguard system for induced pluripotent stem cell-derived rejuvenated T cell therapy

The discovery of induced pluripotent stem cells (iPSCs) has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy

Targeted reversion of induced pluripotent stem cells from patients with human cleidocranial dysplasia improves bone regeneration in a rat calvarial bone defect model

BackgroundRunt-related transcription factor 2 (RUNX2) haploinsufficiency causes cleidocranial dysplasia (CCD) which is characterized by supernumerary teeth, short stature, clavicular dysplasia, and osteoporosis. At present, as a therapeutic strategy for osteoporosis, mesenchymal stem cell (MSC) transplantation therapy is performed in addition to drug therapy. However, MSC-based therapy for osteoporosis in CCD patients is difficult due to a reduction in the ability of MSCs to differentiate into osteoblasts resulting from impaired RUNX2 function. Here, we investigated whether induced pluripotent stem cells (iPSCs) properly differentiate into osteoblasts after repairing the RUNX2 mutation in iPSCs derived from CCD patients to establish normal iPSCs, and whether engraftment of osteoblasts derived from properly reverted iPSCs results in better regeneration in immunodeficient rat calvarial bone defect models.MethodsTwo cases of CCD patient-derived induced pluripotent stem cells (CCD-iPSCs) were generated using retroviral vectors (OCT3/4, SOX2, KLF4, and c-MYC) or a Sendai virus SeVdp vector (KOSM302L). Reverted iPSCs were established using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-derived RNA-guided endonucleases, to correct mutations in CCD-iPSCs. The mRNA expressions of osteoblast-specific markers were analyzed using quantitative reverse-transcriptase polymerase chain reaction. iPSCs-derived osteoblasts were transplanted into rat calvarial bone defects, and bone regeneration was evaluated using microcomputed tomography analysis and histological analysis.ResultsMutation analysis showed that both contained nonsense mutations: one at the very beginning of exon 1 and the other at the initial position of the nuclear matrix-targeting signal. The osteoblasts derived from CCD-iPSCs (CCD-OBs) expressed low levels of several osteoblast differentiation markers, and transplantation of these osteoblasts into calvarial bone defects created in rats with severe combined immunodeficiency showed poor regeneration. However, reverted iPSCs improved the abnormal osteoblast differentiation which resulted in much better engraftment into the rat calvarial bone defect.ConclusionsTaken together, these results demonstrate that patient-specific iPSC technology can not only provide a useful disease model to elucidate the role of RUNX2 in osteoblastic differentiation but also raises the tantalizing prospect that reverted iPSCs might provide a practical medical treatment for CCD

Genetic Variability Overrides the Impact of Parental Cell Type and Determines iPSC Differentiation Potential

Reports on the retention of somatic cell memory in induced pluripotent stem cells (iPSCs) have complicated the selection of the optimal cell type for the generation of iPSC biobanks. To address this issue we compared transcriptomic, epigenetic, and differentiation propensities of genetically matched human iPSCs derived from fibroblasts and blood, two tissues of the most practical relevance for biobanking. Our results show that iPSC lines derived from the same donor are highly similar to each other. However, genetic variation imparts a donor-specific expression and methylation profile in reprogrammed cells that leads to variable functional capacities of iPSC lines. Our results suggest that integration-free, bona fide iPSC lines from fibroblasts and blood can be combined in repositories to form biobanks. Due to the impact of genetic variation on iPSC differentiation, biobanks should contain cells from large numbers of donors.Peer reviewe

Simple and effective generation of transgene-free induced pluripotent stem cells using an auto-erasable Sendai virus vector responding to microRNA-302

Transgene-free induced pluripotent stem cells (iPSCs) are valuable for both basic research and potential clinical applications. We previously reported that a replication-defective and persistent Sendai virus (SeVdp) vector harboring four reprogramming factors (SeVdp-iPS) can efficiently induce generation of transgene-free iPSCs. This vector can express all four factors stably and simultaneously without chromosomal integration and can be eliminated completely from reprogrammed cells by suppressing vector-derived RNA-dependent RNA polymerase. Here, we describe an improved SeVdp-iPS vector (SeVdp(KOSM)302L) that is automatically erased in response to microRNA-302 (miR-302), uniquely expressed in pluripotent stem cells (PSCs). Gene expression and genome replication of the SeVdp-302L vector, which contains miRNA-302a target sequences at the 3′ untranslated region of L mRNA, are strongly suppressed in PSCs. Consequently, SeVdp(KOSM)302L induces expression of reprogramming factors in somatic cells, while it is automatically erased from cells successfully reprogrammed to express miR-302. As this vector can reprogram somatic cells into transgene-free iPSCs without the aid of exogenous short interfering RNA (siRNA), the results we present here demonstrate that this vector may become an invaluable tool for the generation of human iPSCs for future clinical applications

Generation of three induced pluripotent stem cell lines from postmortem tissue derived following sudden death of a young patient with STXBP1 mutation

We established three iPSC lines from postmortem-cultured fibroblasts derived following the sudden unexpected death of an 8-year-old girl with Lennox-Gastaut syndrome, who turned out to have the R551H-mutant STXBP1 gene. These iPSC clones showed pluripotent characteristics while retaining the genotype and demonstrated trilineage differentiation capability, indicating their utility in disease-modeling studies, i.e., STXBP1-encephalopathy. This is the first report on the establishment of iPSCs from a sudden death child, suggesting the possible use of postmortem-iPSC technologies as an epoch-making approach for precise identification of the cause of sudden death

Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

Background: Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool forelucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patientperipheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimuminvasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitorcells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally requireHSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogrammingefficiencies, making the overall procedure costly, laborious, and time-consuming.Methods: We have established a highly efficient method for generating iPSCs from non-mobilized PB-derivedCD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and werekept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads,with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vectorSeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generationseries, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time wererecorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cellreceptor gene rearrangement, pluripotency markers, and differentiation capability were examined.Results：We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay.Conclusion：This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases

Novel Strategy to Control Transgene Expression Mediated by a Sendai Virus-Based Vector Using a Nonstructural C Protein and Endogenous MicroRNAs

<div><p>Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However, existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study, we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because of the capacity of Sendai virus (SeV) nonstructural C proteins to specifically inhibit viral RNA synthesis, overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression, target sequences for an endogenous microRNA were incorporated into the 3′ untranslated region of the <i>C</i> genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore, the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications.</p></div