Disease modeling and drug screening for neurological diseases using human induced pluripotent stem cells

With the general decline of pharmaceutical research productivity, there are concerns that many components of the drug discovery process need to be redesigned and optimized. For example, the human immortalized cell lines or animal primary cells commonly used in traditional drug screening may not faithfully recapitulate the pathological mechanisms of human diseases, leading to biases in assays, targets, or compounds that do not effectively address disease mechanisms. Recent advances in stem cell research, especially in the development of induced pluripotent stem cell (iPSC) technology, provide a new paradigm for drug screening by permitting the use of human cells with the same genetic makeup as the patients without the typical quantity constraints associated with patient primary cells. In this article, we will review the progress made to date on cellular disease models using human stem cells, with a focus on patient-specific iPSCs for neurological diseases. We will discuss the key challenges and the factors that associated with the success of using stem cell models for drug discovery through examples from monogenic diseases, diseases with various known genetic components, and complex diseases caused by a combination of genetic, environmental and other factors.     

Glioma gene therapy using induced pluripotent stem cell derived neural stem cells

Using neural stem cells (NSCs) with tumor tropic migratory capacity to deliver therapeutic genes is an attractive strategy in eliminating metastatic or disseminated tumors. While different methods have been developed to isolate or generate NSCs, it has not been assessed whether induced pluripotent stem (iPS) cells, a type of pluripotent stem cells that hold great potential for regenerative medicine, can be used as a source for derivation of NSCs with tumor tropism. In this study, we used a conventional lentivirus transduction method to derive iPS cells from primary mouse embryonic fibroblasts and then generated NSCs from the iPS cells. To investigate whether the iPS cell derived NSCs can be used in the treatment of disseminated brain tumors, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected into the cerebral hemisphere contralateral to a tumor inoculation site in a mouse intracranial human glioma xenograft model. We observed that NSCs expressing the suicide gene were, in the presence of ganciclovir, effective in inhibiting the growth of the glioma xenografts and prolonging survival of tumor-bearing mice. Our findings provide evidence for the feasibility of using iPS cell derived NSCs as cellular vehicles for targeted anticancer gene therapy.     

Modeling complex neuropsychiatric disorders with human induced pluripotent stem cells

Identifying the molecular and cellular basis of complex neuropsychiatric disorders (cNPDs) has been limited by the inaccessibility of central neurons, variability within broad diagnostic classifications, and the interplay of genetic and environmental factors. Recent work utilizing neuronally differentiated human induced pluripotent stem cells (hiPSCs) from Mendelian and polygenic cNPDs is beginning to illuminate neuritic, synaptic or cell body variations accompanied by specific gene or protein expression alterations largely mimicking known pathology. In some cases, phenotypes have only emerged after application of cellular stress or long duration of differentiation. Pathological and cellular expression features are fully or partially responsive to pharmacological treatment highlighting the potential utility of differentiated hiPSCs for discovery of personalized therapeutics and for identifying pathogenetically relevant targets in subgroups of patients within a broad syndromic classification. Because of the inherent variability in developing and differentiating hiPSC lines and the multiple comparisons implicit in 'omics' technologies, rigorous algorithms for assuring statistical significance and independent confirmation of results, will be required for robust modeling of cNPDs.     

Generation of functional endothelial cells with progenitor-like features from murine induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) have shown great potential in regenerative medicine and research applications like disease modeling or drug discovery. Endothelium is indispensable for vascular homeostasis, whereas endothelial dysfunction could lead to different diseases. Therefore, generating autologous cells, able to restore the endothelial lining, can be crucial for slowing or reversing certain pathological processes. In the current study we show efficient differentiation of murine iPSCs into endothelial cells (ECs) with stable CD34 +/Tie-2 +/Sca-1 +/CD45 − phenotype and proven functionality. iPS-derived ECs (iPS-ECs) were positive for phospho-eNOS and von Willebrand factor, and responded to shear stress with up-regulation of KLF-2, KDR, HO-1, and increased nitric oxide and VEGF production. These cells reacted to cytokine stimulation through increase in VCAM-1 and inflammatory cytokine secretion. iPS-ECs showed also certain progenitor features, like expression of progenitor markers (CD34, Sca-1, c-kit) and high clonogenic potential. The angiogenic capacity of iPS-ECs in spheroid sprouting assay was similar to primary ECs, whereas on Matrigel, tube structures could be formed only in the presence of other support cells. Angiogenic potential of iPS-ECs in vivo, was similar to murine endothelial cell line MS-1. Summarizing, our approach enabled generation of functional progenitor-like ECs, which can be used as a research model.     

Proarrhythmia risk prediction using human induced pluripotent stem cell-derived cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are expected to become a useful tool for proarrhythmia risk prediction in the non-clinical drug development phase. Several features including electrophysiological properties, ion channel expression profile and drug responses were investigated using commercially available hiPSC-CMs, such as iCell-CMs and Cor.4U-CMs. Although drug-induced arrhythmia has been extensively examined by microelectrode array (MEA) assays in iCell-CMs, it has not been fully understood an availability of Cor.4U-CMs for proarrhythmia risk. Here, we evaluated the predictivity of proarrhythmia risk using Cor.4U-CMs. MEA assay revealed linear regression between inter-spike interval and field potential duration (FPD). The hERG inhibitor E?4031 induced reverse-use dependent FPD prolongation. We next evaluated the proarrhythmia risk prediction by a two-dimensional map, which we have previously proposed. We determined the relative torsade de pointes risk score, based on the extent of FPD with Fridericia's correction (FPDcF) change and early afterdepolarization occurrence, and calculated the margins normalized to free effective therapeutic plasma concentrations. The drugs were classified into three risk groups using the two-dimensional map. This risk-categorization system showed high concordance with the torsadogenic information obtained by a public database CredibleMeds. Taken together, these results indicate that Cor.4U-CMs can be used for drug-induced proarrhythmia risk prediction.     

Silver nanoparticles inhibit neural induction in human induced pluripotent stem cells

Abstract

Silver nanoparticles (AgNPs) have been widely used as consumer products due to their antibacterial activities. Despite their extensive use, AgNPs have been reported to cause various types of cytotoxicity, including neurotoxicity. However, the potential action of AgNPs on early fetal development has not been elucidated. This study determined the effects of AgNPs on neural induction in human induced pluripotent stem cells (iPSCs), used as a model for human fetal stage development. It was observed that exposure to AgNPs reduced the expression of several neural differentiation marker genes, including OTX2, an early biomarker for neurogenesis in iPSCs. Since neural differentiation requires ATP as a source of energy, the intracellular ATP content was also measured. It was observed that AgNPs decreased intracellular ATP levels in iPSCs. Since AgNPs suppressed energy production, a critical mitochondrial function, the effects of AgNPs on mitochondrial dynamics were further studied. The results revealed that AgNPs induced mitochondrial fragmentation and reduced the level of mitochondrial fusion protein mitofusin 1 (Mfn1). Previously, we reported that knockdown of Mfn1 in iPSCs inhibited neural induction via OTX2 downregulation. This suggested that AgNPs could induce cytotoxicity, including neurodevelopmental toxicity, via Mfn1-mediated mitochondrial dysfunction in iPSCs. Thus, mitochondrial function in iPSCs can be used for assessing the cytotoxic effects associated with nanomaterials, including AgNPs.     

Electrophysiological characterization of cardiomyocytes derived from human induced pluripotent stem cells

Cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs) hold great promise for development of in vitro research tools to assess cardiotoxicity, including QT prolongation. In the present study, we aimed to clarify the electrophysiological/pharmacological characteristics of hiPS-CMs using the patch-clamp technique. The hiPS cells were differentiated into beating cardiomyocytes by the embryoid body method. The expression of genes related to cardiac ion channels and differentiation markers in cardiomyocytes were detected by RT-PCR. Whole-cell patch-clamp recordings were performed using single hiPS-CMs dispersed from beating colonies. We confirmed voltage-dependence of major cardiac ion currents (I(Na), I(Ca), I(Kr), and I(Ks)) and pharmacological responses to ion-channel blockers. Action potential duration (APD) was prolonged by both I(Kr)/hERG and I(Ks) blockers, whereas it was shortened by an I(Ca) blocker, indicating that these ion current components contribute to action potential generation in hiPS-CMs. As for multiple ion channel blockers, terfenadine prolonged APD, but verapamil did not, results which were identical to clinically relevant pharmacological responses. These data suggest that patch-clamp assay using hiPS-CMs could be an accurate method of predicting the human cardiac responses to drug candidates. This study would be helpful in establishing an electrophysiological assay to assess the risk of drug-induced arrhythmia using hiPS-CMs.     

Application of human pluripotent stem cells and pluripotent stem cell-derived cellular models for assessing drug toxicity

Introduction: Human pluripotent stem cells (hPSCs) are capable of differentiating into all types of cells in the body and so provide suitable toxicology screening systems even for hard-to-obtain human tissues. Since hPSCs can also be generated from differentiated cells and current gene editing technologies allow targeted genome modifications, hPSCs can be applied for drug toxicity screening both in normal and disease-specific models. Targeted hPSC differentiation is still a challenge but cardiac, neuronal or liver cells, and complex cellular models are already available for practical applications.

Areas covered: The authors review new gene-editing and cell-biology technologies to generate sensitive toxicity screening systems based on hPSCs. Then the authors present the use of undifferentiated hPSCs for examining embryonic toxicity and discuss drug screening possibilities in hPSC-derived models. The authors focus on the application of human cardiomyocytes, hepatocytes, and neural cultures in toxicity testing, and discuss the recent possibilities for drug screening in a ‘body-on-a-chip’ model system.

Expert opinion: hPSCs and their genetically engineered derivatives provide new possibilities to investigate drug toxicity in human tissues. The key issues in this regard are still the selection and generation of proper model systems, and the interpretation of the results in understanding in vivo drug effects.     

Optimization of adenovirus vectors for transduction in embryonic stem cells and induced pluripotent stem cells

Because embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can differentiate into various types of cells in vitro, they are considered as a valuable model to understand the processes involved in the differentiation into functional cells as well as an unlimited source of cells for therapeutic applications. Efficient gene transduction method is one of the powerful tools for the basic researches and for differentiating ES and iPS cells into lineage-committed cells. Recently, we have developed an adenovirus (Ad) vector for efficient transduction into ES and iPS cells. We showed that Ad vectors containing the cytomegalovirus enhancer/β-actin promoter with β-actin intron (CA) promoter or the elongation factor (EF)-1α promoter were the appropriate for the transduction into ES and iPS cells. We also found that enforced expression of a PPARγ gene or a Runx2 gene into mouse ES and iPS cells by an optimized Ad vector markedly augmented the differentiation of adipocytes or osteoblasts, respectively. Thus, a gene transfer technique using an Ad vector could be an advantage for the regulation of stem cell differentiation and could be applied to regenerative medicine based on ES and iPS cells.     

人诱导多能干细胞在药物早期毒性评价中的应用

人诱导多能干细胞与人胚胎干细胞相似,在体外可分化为各种类型的体细胞,其来源充足,可针对个人或某种疾病取材,为药物早期毒性评价体外替代方法提供了一个新的可选细胞模型。目前,利用人诱导多能干细胞获得的心肌细胞用于药物引起的心率改变、QT间期延长和心肌损伤等心脏毒性评价;利用其获得的神经细胞,结合高通量、高内涵技术及电生理学技术,可用于药物引起的神经突出生长异常、电生理改变及神经发育毒性评价;利用人诱导多能干细胞可获得个体特异性的大量肝细胞,具有较高的细胞色素P450酶活性,能够比较真实的反映人肝细胞的代谢特征,用于评价药物肝细胞毒性;人诱导多能干细胞具有多能性,在体外可分化为外、中和内胚层,具有用于发育毒性评价的可能性,对三胚层相应标志分子的检测有助于发育毒性评价终点的确立;人诱导多能干细胞还可用作3D培养的种子细胞,构建三维立体组织和器官模型,用于药物早期毒性评价,进一步缩小细胞水平与体内水平评价结果的差异。     

诱导多能干细胞来源间充质干细胞对大鼠骨关节炎的治疗作用

目的 研究诱导多能干细胞来源间充质干细胞(iPSC-MSCs)对大鼠骨关节炎(OA)的治疗作用。方法 采用Hulth法建立大鼠OA模型,编号后运用Excel表格随机分组功能将其分为模型组、iPSC-MSCs组、人脐带来源间充质干细胞(hUC-MSCs)组、玻璃酸钠(HA)阳性对照组,另设假手术组作为对照,每组10只。除HA组动物多次给药(每周1次,共给药5次)外,其它各组均单次给药,每次每只动物在术侧关节腔注射50 μL药物iPSC-MSCs和hUC-MSCs,假手术组和模型组同法给予生理盐水。给药5周后处死动物,观察iPSC-MSCs对关节直径差值、关节腔结构及评分、关节面评分、病理学评分等指标的影响;检测膝关节基质金属蛋白酶(MMP)-13、Ⅱ型胶原(CollagenⅡ)及解聚蛋白样金属蛋白酶(ADAMTS)-5表达情况。结果 iPSC-MSCs和hUC-MSCs给药均可显著降低膝关节面Pelletier评分、关节直径和病理Mankin评分,改善关节腔结构,降低X线评分和关节肿胀,且iPSC-MSCs较hUC-MSCs给药在降低Pelletier评分[(1.22±0.67)比(2.00±0.71),P=0.021]方面差异有统计学意义;相比于模型组,iPSC-MSCs关节腔注射给药可显著增加软骨层CollagenⅡ表达[(35.87±8.52)比(69.90±7.65),P＜0.01],降低MMP-13[(59.25±5.56)比(33.75±5.85),P＜0.01]和ADAMTS-5[(52.25±10.47)比(22.25±6.13),P＜0.01]表达水平,且与hUC-MSCs给药差异有统计学意义,iPSC-MSCs组与hUC-MSCs组CollagenⅡ、MMP-13、ADAMTS-5值分别为[(69.90±7.65)比(61.00±5.52),P=0.010]、[(33.75±5.85)比(43.25±6.02),P=0.028]、[(22.25±6.13)比(36.50±4.65),P=0.011]。结论 关节腔注射iPSC-MSCs可显著改善OA大鼠关节病理,具有明显的软骨保护作用。     

Pharmacokinetic functions of human induced pluripotent stem cell-derived small intestinal epithelial cells

To develop a novel intestinal drug absorption system using intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells, the cells must possess sufficient pharmacokinetic functions. However, the CYP3A4/5 activities of human iPS cell-derived small intestinal epithelial cells prepared using conventional differentiation methods is low. Further, studies of the CYP3A4/5 activities of human iPS-derived and primary small intestinal cells are not available. To fill this gap in our knowledge, here we used forskolin to develop a new differentiation protocol that activates adenosine monophosphate signaling. mRNA expressions of human iPS cell-derived small intestinal epithelial cells, such as small intestine markers, drug-metabolizing enzymes, and drug transporters, were comparable to or greater than those of the adult small intestine. The activities of CYP3A4/5 in the differentiated cells were equal to those of human primary small intestinal cells. The differentiated cells had P-glycoprotein and PEPT1 activities equivalent to those of Caco-2 cells. Differentiated cells were superior to Caco-2 cells for predicting the membrane permeability of drugs that were absorbed through a paracellular pathway and via drug transporters. In summary, here we produced human iPS cell-derived small intestinal epithelial cells with CYP3A4/5 activities equivalent to those of human primary small intestinal cells.     

利用诱导多能干细胞构建流产毒性药物的体外筛查模型

妊娠期用药不可避免,如何帮助孕妇合理用药以减少自发流产、早产和低体重儿的发生,对于医务工作者至关重要.本研究建立了药物流产毒性的检测模型,可筛除疑似流产的药物,避免用药不当导致的孕妇流产.通过三维悬浮培养诱导多能干细胞(induced pluripotent stem cells,iPSCs)的方式,诱导其形成拟胚体(...     

诱导性多能干细胞的研究进展

诱导性多能干细胞(induced pluripotent stem cell,iPSC)是通过将几个特定的转录因子导入分化的体细胞以诱导体细胞重编程而获得的可不断自我更新且具有多向分化潜能的多能干细胞.如同胚胎干细胞,iPSC仍维持未分化状态,可分化为3个胚层来源的所有细胞以参与机体所有组织和器官的形成.这项技术避免了干细胞研究领域的免疫排斥和伦理道德问题,同时为特异性疾病的研究提供了强有力的工具.因此,iPSC研究不仅具有重要的理论研究意义,而且在再生医学及药物筛选与评价方面具有很好的应用价值.