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Development of selective cytotoxic viral vectors for concentration of undifferentiated cells in cardiomyocytes derived from human induced pluripotent stem cells.

ABSTRACT: Cell-processed therapeutic products (CTPs) derived from human pluripotent stem cells (hPSCs) have innovative applications in regenerative medicine. However, undifferentiated hPSCs possess tumorigenic potential; thus, sensitive methods for the detection of residual undifferentiated hPSCs are essential for the clinical use of hPSC-derived CTPs. The detection limit of the methods currently available is 1/10⁵ (0.001%, undifferentiated hPSCs/differentiated cells) or more, which could be insufficient for the detection of residual hPSCs when CTPs contain more than 1 × 10⁵ cells. In this study, we developed a novel approach to overcome this challenge, using adenovirus and adeno-associated virus (AdV and AAV)-based selective cytotoxic vectors. We constructed AdV and AAV vectors that possess a suicide gene, iCaspase 9 (iCasp9), regulated by the CMV promoter, which is dormant in hPSCs, for the selective expression of iCasp9 in differentiated cells. As expected, AdV/CMV-iCasp9 and AAV/CMV-iCasp9 exhibited cytotoxicity in cardiomyocytes but not in human induced pluripotent stem cells (hiPSCs). The vectors also induced apoptosis in hiPSC-derived cardiomyocytes, and the surviving cells exhibited higher levels of hPSC marker expression. These results indicate that the AdV- and AAV-based cytotoxic vectors concentrate cells expressing the undifferentiated cell markers in hiPSC-derived products and are promising biological tools for verifying the quality of CTPs.

SUBMITTER: Kono K

PROVIDER: S-EPMC6403330 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Development of selective cytotoxic viral vectors for concentration of undifferentiated cells in cardiomyocytes derived from human induced pluripotent stem cells.

Kono Ken K Sawada Rumi R Kuroda Takuya T Yasuda Satoshi S Matsuyama Satoko S Matsuyama Akifumi A Mizuguchi Hiroyuki H Sato Yoji Y

Scientific reports 20190306 1

Cell-processed therapeutic products (CTPs) derived from human pluripotent stem cells (hPSCs) have innovative applications in regenerative medicine. However, undifferentiated hPSCs possess tumorigenic potential; thus, sensitive methods for the detection of residual undifferentiated hPSCs are essential for the clinical use of hPSC-derived CTPs. The detection limit of the methods currently available is 1/10<sup>5</sup> (0.001%, undifferentiated hPSCs/differentiated cells) or more, which could be in ...[more]

PMID: 30842516

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Project description:BackgroundPluripotent stem cells (PSCs) such as embryonic stem cells and induced pluripotent stem cells are promising target cells for cell regenerative medicine together with recently advanced technology of in-vitro differentiation. However, residual undifferentiated stem cells (USCs) during in-vitro differentiation are considered a potential risk for development of cancer cells and nonspecific lineage cell types. In this study we observed that USCs still exist during hepatic differentiation, consequently resulting in poor quality of the hepatic population and forming teratoma in vivo. Therefore, we hypothesized that effectively removing USCs from in-vitro differentiation could improve the quality of the hepatic population and guarantee safety from risk of teratoma formation.MethodsHuman PSCs were differentiated to hepatocytes via four steps. YM155, a known BIRC5 inhibitor, was applied for removing the residual USCs on the hepatic differentiation. After YM155 treatment, hepatocyte development was evaluated by measuring gene expression, immunostaining and hepatic functions at each stage of differentiation, and forming teratomas were confirmed by cell transplantation with or without YM155.ResultsThe selected concentrations of YM155 removed USCs (NANOG+ and OCT4+) in a dose-dependent manner. As a result, expression of endodermal markers (SOX17, FOXA2 and CXCR4) at stage II of differentiation and hepatic markers (ALB, AFP and HNF4A) at stage III was up-regulated by YM155 treatment as well as the hepatic population (ALB+), and functions (ALB/urea secretion and CYP450 enzyme activity) were enhanced at the final stage of differentiation (stage IV). Furthermore, we demonstrated that NANOG and OCT4 expression remaining until stage III (day 15 of differentiation) completely disappeared when treated with YM155 and teratoma formation was effectively prevented by YM155 pretreatment in the in-vitro study.ConclusionsWe suggest that the removal of USCs using YM155 could improve the quantity and quality of induced hepatocytes and eliminate the potential risk of teratoma formation.

Project description:Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte (CM) phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived CMs would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early-stage counterparts (PSC-CMs after 20-40 days of in vitro differentiation and culture), late-stage hESC-CMs and hiPSC-CMs (80-120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by bright-field microscopy, and a 10-fold increase in the fraction of multinucleated CMs. Ultrastructural analysis confirmed improvements in the myofibrillar density, alignment, and morphology. We measured the contractile performance of late-stage hESC-CMs and hiPSC-CMs and noted a doubling in shortening magnitude with slowed contraction kinetics compared to the early-stage cells. We then examined changes in the calcium-handling properties of these matured CMs and found an increase in calcium release and reuptake rates with no change in the maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found that late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of the key cardiac structural markers, including ?-myosin heavy chain and connexin-43, in late-stage hESC-CMs and hiPSC-CMs. These findings suggest that PSC-CMs are capable of slowly maturing to more closely resemble the phenotype of adult CMs and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue.

Dataset Information

Development of selective cytotoxic viral vectors for concentration of undifferentiated cells in cardiomyocytes derived from human induced pluripotent stem cells.

Publications

Development of selective cytotoxic viral vectors for concentration of undifferentiated cells in cardiomyocytes derived from human induced pluripotent stem cells.

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