A comparative study of neural and mesenchymal stem cell-based carriers for oncolytic adenovirus in a model of malignant glioma

Glioblastoma multiforme is a primary malignancy of the central nervous system that is universally fatal due to its disseminated nature. Recent investigations have focused on the unique tumor-tropic properties of stem cells as a novel platform for targeted delivery of anticancer agents to the brain. Neural stem cells (NSCs) and mesenchymal stem cells (MSCs) both have the potential to function as cell carriers for targeted delivery of a glioma restricted oncolytic virus to disseminated tumor due to their reported tumor tropism. In this study, we evaluated NSCs and MSCs as cellular delivery vehicles for an oncolytic adenovirus in the context of human glioma. We report the first preclinical comparison of the two cell lines and show that, while both stem cell lines are able to support therapeutic adenoviral replication intracellularly, the amount of virus released from NSCs was a log higher than the MSC (p < 0.001). Moreover, only virus loaded NSCs that were administered intracranially in an orthotopic glioma model significantly prolonged the survival of tumor bearing animals (median survival for NSCs 68.5 days vs 44 days for MSCs, p < 0.002). Loading oncolytic adenovirus into NSCs and MSCs also led to expression of both pro- and anti-inflammatory genes and decreased vector-mediated neuroinflammation. Our results indicate that, despite possessing a comparable migratory capacity, NSCs display superior therapeutic efficacy in the context of intracranial tumors. Taken together, these findings argue in favor of NSCs as an effective cell carrier for antiglioma oncolytic virotherapy.     

Establishment of MDR1-knockout human induced pluripotent stem cell line

Multiple drug resistance 1 (MDR1) is highly expressed in various organs, including the liver, small intestine, and blood–brain barrier (BBB). Because MDR1 plays important roles in the excretion of many drugs, it is necessary to evaluate whether drug candidates are potential substrates of MDR1. Recently, many researchers have shown that human induced pluripotent stem (iPS) cell-derived differentiated cells such as hepatocytes and enterocytes can be applied for pharmacokinetic testing. Here, we attempted to generate MDR1-knockout (KO) iPS cell lines using genome editing technology. The correctly targeted human iPS cell lines were successfully obtained. The expression levels of pluripotent markers in human iPS cells were not changed by MDR1 knockout. The gene expression levels of hepatic markers in MDR1-KO iPS-derived hepatocyte-like cells were higher than those in undifferentiated MDR1-KO iPS cells, suggesting that MDR1-KO iPS cells have hepatic differentiation capacity. In addition, MDR1 expression levels were hardly detected in MDR1-KO iPS cell–derived hepatocyte-like cells. We thus succeeded in establishing MDR1-KO iPS cell lines that could be utilized for pharmacokinetic testing.     

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.     

Induced pluripotent stem cell research: A revolutionary approach to face the challenges in drug screening

Discovery of induced pluripotent stem (iPS) cells in 2006 provided a new path for cell transplantation and drug screening. The iPS cells are stem cells derived from somatic cells that have been genetically reprogrammed into a pluripotent state. Similar to embryonic stem (ES) cells, iPS cells are capable of differentiating into three germ layers, eliminating some of the hurdles in ES cell technology. Further progress and advances in iPS cell technology, from viral to non-viral systems and from integrating to non-integrating approaches of foreign genes into the host genome, have enhanced the existing technology, making it more feasible for clinical applications. In particular, advances in iPS cell technology should enable autologous transplantation and more efficient drug discovery. Cell transplantation may lead to improved treatments for various diseases, including neurological, endocrine, and hepatic diseases. In studies on drug discovery, iPS cells generated from patient-derived somatic cells could be differentiated into specific cells expressing specific phenotypes, which could then be used as disease models. Thus, iPS cells can be helpful in understanding the mechanisms of disease progression and in cell-based efficient drug screening. Here, we summarize the history and progress of iPS cell technology, provide support for the growing interest in iPS cell applications with emphasis on practical uses in cell-based drug screening, and discuss some challenges faced in the use of this technology.     

Differentiation of functional cells from iPS cells by efficient gene transfer

Induced pluripotent stem (iPS) cells, which are generated from somatic cells by transducing four genes, are expected to have broad application to regenerative medicine. Although establishment of an efficient gene transfer system for iPS cells is considered to be essential for differentiating them into functional cells, the detailed transduction characteristics of iPS cells have not been examined. By using an adenovirus (Ad) vector containing the cytomegalovirus enhancer/beta-actin (CA) promoters, we have developed an efficient transduction system for mouse mesenchymal stem cells and embryonic stem (ES) cells. Also, we applied our transduction system to mouse iPS cells and investigated whether efficient differentiation could be achieved by Ad vector-mediated transduction of a functional gene. As in the case of ES cells, the Ad vector could efficiently transduce transgenes into mouse iPS cells. We found that the CA promoter had potent transduction ability in iPS cells. Moreover, exogenous expression of a PPARγ gene or a Runx2 gene into mouse iPS cells by an optimized Ad vector enhanced adipocyte or osteoblast differentiation, respectively. These results suggest that Ad vector-mediated transient transduction is sufficient to promote cellular differentiation and that our transduction methods would be useful for therapeutic applications based on iPS cells.     

诱导多潜能干细胞在临床治疗中的研究进展及前景分析

近年来,不同的研究组先后证实:通过转染转录因子组合可诱导终末分化的细胞重新编程为多潜能状态,由此获得的细胞无论是在表观遗传,还是在分化潜能上都与胚胎干细胞十分相似,因此被称为诱导多潜能干细胞。iPS细胞具有能分化成为三个胚层全部种类细胞的能力,并且其细胞来源不受伦理限制,所以在细胞治疗、组织工程和药物开发等领域被寄予厚望。本文综述了iPS细胞安全制备和临床应用的相关研究进展。最新的研究成果预示着不久的将来,我们能够更加高效、安全地建立疾病特异性或个体特异性iPS细胞系,这无疑会掀起一场再生医学的革命。     

PPARβ/δ-agonist F3SM and GW501516 ameliorates dysfunction in glycolysis in sAD patient iPSCs derived neural stem cells

OBJECTIVE To establish an in vitro cell model based on sAD patient-specific human neural stem cells and explore the energy metabolism of the neural stem cel s(NSCs), then observe whether PPARβ/δ-agonist F3SM and GW501516 can improve the energy metabolism of NSCs. METHODS The induced pluripotent stem cells(i PSCs) of sAD patients and healthy controls were first induced into neural stem cells. Then the differentiated NSCs were identified by immunofluorescence.The energy metabolism of NSCs was detected by Seahorse XF96 Extracellular Flux Analyzer(Seahorse Bioscience) and the rate of the extracellular pH drift(ECAR) was analyzed, which can reflect the glycolysis level of cells.RESULTS NSCs derived from sAD patients and healthy controls expressed neural stem cell markers Nestin,Sox1, Sox2 and Ki67. The analysis of ECAR showed that the basic glycolysis level and the maximum glycolysis capacity of the NSCs from healthy controls were significantly higher than those of sAD patients, which reflected abnormal energy metabolism of NSCs derived from sAD patients. While PPARβ/δ-agonist F3 SM and GW501516 can ameliorate dysfunction in glycolysisin sAD patient NSCs by enhancing the level of basic glycolysis and maximum glycolysis capacity. CONCLUSION On the basis of the experiments we conducted, we can conclude that sADi PSCs-derived NSCs exhibited dysfunction in energy metabolism, revealing the pathological characteristics of early sAD. PPARβ/δ-agonist F3SM and GW501516 can ameliorate dysfunctionin glycolysisin sAD patient NSCs,which might be served as new therapeutic strategy for AD.     

Cardiac safety pharmacology: from human ether-a-gogo related gene channel block towards induced pluripotent stem cell based disease models

INTRODUCTION: The field of cardiac safety pharmacology has been experiencing exciting changes over the recent years. Drug induced arrhythmia of the torsade des pointes types has been the reason for the denial of approval of novel drug candidates. The aim of cardiac safety pharmacology is to detect undesirable pharmacodynamic drug effects within and above the therapeutic range. A special focus is on the identification of potential arrhythmogenic effects within the drug discovery chain. AREAS COVERED: Here, the authors discuss the relevance of induced pluripotent stem (iPS) cell derived cardiomyocytes for safety pharmacology. The technology of obtaining functional cardiomyocytes from somatic cells of healthy donors and patients with inherited diseases is the basis for diverse disease models in multi-level safety pharmacology screening. The reader will gain an overview of stem cell based technologies in cardiac safety pharmacology in cardiac and disease modeling by iPS cell derived cardiomyocytes from patients with an inherited cardiac syndrome. EXPERT OPINION: iPS cell derived cardiomyocytes - especially from patients with increased risk of cardiac arrhythmia - are on the verge of offering new options for drug testing. More reliable assays can be expected to predict the arrhythmogenic risk of drug candidates in humans. However, this technology is still new and extensive validation studies are due.     

Induced pluripotent stem (iPS) cells repair and regenerate infarcted myocardium

Cardiac myocyte differentiation reported thus far is from iPS cells generated from mouse and human fibroblasts. However, there is no article on the generation of iPS cells from cardiac ventricular specific cell types such as H9c2 cells. Therefore, whether transduced H9c2 cells, originally isolated from embryonic cardiac ventricular tissue, will be able to generate iPS cells and have the potential to repair and regenerate infarcted myocardium remains completely elusive. We transduced H9c2 cells with four stemness factors, Oct3/4, Sox2, Klf4, and c-Myc, and successfully reprogrammed them into iPS cells. These iPS cells were able to differentiate into beating cardiac myocytes and positively stained for cardiac specific sarcomeric α-actin and myosin heavy chain proteins. Following transplantation in the infarcted myocardium, there were newly differentiated cardiac myocytes and formation of gap junction proteins at 2 weeks post-myocardial infarction (MI), suggesting newly formed cardiac myocytes were integrated into the native myocardium. Furthermore, transplanted iPS cells significantly (p < 0.05) inhibited apoptosis and fibrosis and improved cardiac function compared with MI and MI+H9c2 cell groups. Moreover, our iPS cell derived cardiac myocyte differentiation in vitro and in vivo was comparable to embryonic stem cells in the present study. In conclusion we report for the first time that we have H9c2 cell-derived iPS cells which contain the potential to differentiate into cardiac myocytes in the cell culture system and repair and regenerate infarcted myocardium with improved cardiac function in vivo.     

胶质瘤细胞诱导神经干细胞迁移作用的实验研究

目的观察胶质瘤细胞在体外培养条件下对神经干细胞是否有诱导迁移的作用。方法①从新生1～2dSD大鼠脑皮层分离培养神经干细胞,进行神经干细胞及其增殖能力鉴定。②胶质瘤细胞与神经干细胞限定区域联合培养,观察神经干细胞的生长及其形态学变化。结果①所获得神经细胞球Nestin染色阳性,传代神经细胞球抗BrdU染色阳性。②可观察到神经干细胞球周围长出细胞突起,在靠近胶质瘤细胞的一侧,细胞突起的密度及长度均大于其他方向上的突起并且可见部分干细胞向胶质瘤细胞方向的移动。结论胶质瘤细胞在体外对神经干细胞有诱导迁移作用。     

Delivery of nucleic acid therapeutics by genetically engineered hematopoietic stem cells

Several populations of adult human stem cells have been identified, but only a few of these are in routine clinical use. The hematopoietic stem cell (HSC) is arguably the most well characterized and the most routinely transplanted adult stem cell. Although details regarding several aspects of this cell's phenotype are not well understood, transplant of HSCs has advanced to become the standard of care for the treatment of a range of monogenic diseases and several types of cancer. It has also proven to be an excellent target for genetic manipulation, and clinical trials have already demonstrated the usefulness of targeting this cell as a means of delivering nucleic acid therapeutics for the treatment of several previously incurable diseases. It is anticipated that additional clinical trials will soon follow, such as genetically engineering HSCs with vectors to treat monogenic diseases such as hemophilia A. In addition to the direct targeting of HSCs, induced pluripotent stem (iPS) cells have the potential to replace virtually any engineered stem cell therapeutic, including HSCs. We now know that for the broad use of genetically modified HSCs for the treatment of non-lethal diseases, e.g. hemophilia A, we must be able to regulate the introduction of nucleic acid sequences into these target cells. We can begin to refine transduction protocols to provide safer approaches to genetically manipulate HSCs and strategies are being developed to improve the overall safety of gene transfer. This review focuses on recent advances in the systemic delivery of nucleic acid therapeutics using genetically modified stem cells, specifically focusing on i) the use of retroviral vectors to genetically modify HSCs, ii) the expression of fVIII from hematopoietic stem cells for the treatment of hemophilia A, and iii) the use of genetically engineered hematopoietic cells generated from iPS cells as treatment for disorders of hematopoiesis.     

胶质瘤干细胞的研究进展

神经胶质瘤是中枢神经系统肿瘤中最常见的、发病率最高的原发性恶性肿瘤,严重威胁人类健康。随着研究的不断深入,人们对胶质瘤也有了更深刻的认识。本文对胶质瘤干细胞理论的研究进展及其对临床实际工作的重要指导意义进行综述。     

Cardiac safety pharmacology: from human ether-a-gogo related gene channel block towards induced pluripotent stem cell based disease models

Introduction: The field of cardiac safety pharmacology has been experiencing exciting changes over the recent years. Drug induced arrhythmia of the torsade des pointes types has been the reason for the denial of approval of novel drug candidates. The aim of cardiac safety pharmacology is to detect undesirable pharmacodynamic drug effects within and above the therapeutic range. A special focus is on the identification of potential arrhythmogenic effects within the drug discovery chain.

Areas covered: Here, the authors discuss the relevance of induced pluripotent stem (iPS) cell derived cardiomyocytes for safety pharmacology. The technology of obtaining functional cardiomyocytes from somatic cells of healthy donors and patients with inherited diseases is the basis for diverse disease models in multi-level safety pharmacology screening. The reader will gain an overview of stem cell based technologies in cardiac safety pharmacology in cardiac and disease modeling by iPS cell derived cardiomyocytes from patients with an inherited cardiac syndrome.

Expert opinion: iPS cell derived cardiomyocytes – especially from patients with increased risk of cardiac arrhythmia – are on the verge of offering new options for drug testing. More reliable assays can be expected to predict the arrhythmogenic risk of drug candidates in humans. However, this technology is still new and extensive validation studies are due.     

Focus on germ-layer markers: A human stem cell-based model for in vitro teratogenicity testing

Human induced pluripotent stem cells (hiPSC) were used to develop an assay format that may deliver information on teratogenicity of drugs. A human pluripotent stem cell scorecard panel was used to monitor the expression of 96 marker genes that are indicative of the stem cell state or differentiation into the ectoderm, mesoderm and endoderm lineages. We selected a human episomal iPS cell line for the assay based on karyotype stability, initial pluripotency, differentiation capacity and overall gene expression variability. The assay is based on embryoid body formation and was developed to be simply automated. In this proof of concept study, we used eight reference compounds (valproic acid, all-trans-retinoic acid, thalidomide, methotrexate, hydroxyurea, ascorbic acid, penicillin G and ibuprofen) to test the technical performance of the assay (readout stability) in concentration-response and time-course experiments. We also found that each compound affected marker gene expression in a different way. Various forms of data analysis identified 19 out of 96 early developmental genes as potential predictive markers for teratogenicity. Machine-learning models were run to exemplify how the assay will be developed further. The preliminary results from these analyses suggest that the assay could be suitable for the pre-screening of candidate pharmaceutical compounds. The approach presented here points a way towards development of a human cell-based assay that could replace the murine EST currently used to screen for early indications of potential teratogenicity of drug candidates.     

Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening

Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs.     

诱导性多能干细胞体外诱导的现状和展望

鉴于人的胚胎干细胞在再生医学、组织工程学和药物研发等领域有极高的应用价值,科学家尝试通过各种途径获得胚胎干细胞样的多能干细胞。其中Yamanaka等率先在体外通过病毒载体诱导的方式实现体细胞重新编程,由此得到诱导性多能干细胞。多种无遗传修饰的诱导方式正在尝试和改进中,例如用小分子化合物来代替外源基因进行重编程引起了很大的兴趣。用基于细胞水平的表型筛选法和信号通路筛选法,已筛选出特异小分子或天然产物,也可以特异地将成熟细胞去分化为干细胞,有望运用于组织修复和再生。而利用重组蛋白在体外将体细胞诱导为干细胞,也获得了初步成功。由诱导性多能干细胞在体外诱导分化出的细胞在治疗相应疾病方面展示出一定疗效。不同类型的干细胞向体细胞的定向分化策略都是基于目前对发育生物学的认识,这些研究揭示了一些共同的可能线索。     

The future of induced pluripotent stem cells for cardiac therapy and drug development

The field of stem cell research was revolutionized with the advent of induced pluripotent stem cells. By reprogramming somatic cells to pluripotent stem cells, most ethical concerns associated with the use of embryonic stem cells are overcome, such that many hopes from the stem cell field now seem a step closer to reality. Several methods and cell sources have been described to create induced pluripotent stem cells and we discuss their characteristics in terms of feasibility and efficiency. From these cells, cardiac progenitors and cardiomyocytes can be derived by several protocols and most recent advances as well as remaining limitations are being discussed. However, in the short time period this technology has been around, evidence emerges that induced pluripotent stem cells may be more prone to genetic defects and maintain an epigenetic memory and thus may not be entirely the same as embryonic stem cells. Despite the lack of a complete fundamental understanding of stem cell biology, and even more of ways how to coax them into defined cell types, the technology is quickly adopted by industry. This paper gives an overview of the current applications of induced pluripotent stem cells in cardiovascular drug development and highlights active areas of research towards functional repair of the damaged heart. Adult stem cells have already been taken to clinical trials and we discuss these results in light of potential and hurdles to be taken to move induced pluripotent stem cells to the clinic.     

Gene therapy with vector-producing multipotent mesenchymal stromal cells

Suicide gene therapy with retroviral vector-producing cells was feasible as an adjuvant to the surgical resection of recurrent glioblastoma, although any benefit appeared to be marginal. Further evaluation of the therapeutic strategy with the vector-producing cells must incorporate improved delivery of vectors and transgenes to the target cells. We have previously demonstrated the ability of vector-producing tumor cells engineered by the adenovirus-retrovirus hybrid vector to destroy satellite tumor cells, although therapeutic efficacy for aggressive tumor has to be further evaluated by the systemic delivery of the vector-producing cells. Mesenchymal stem cells (MSCs) should be an effective delivery vehicle to seek out tumor cells in vivo and transport cancer-killing gene or immune products with minimal rejection reaction by the host. We developed vector-producing tumor-tracking cells to improve suicide cancer gene therapy. Nucleofection was attempted to deliver retrovirus vector components into rodent MSCs. Athymic nude mice with subcutaneous 9L glioma were received vector-producing MSCs through the left ventricular cavity. Optical bioluminescence imaging in vivo revealed accumulation of the MSCs into the subcutaneous 9L tumors but not Rat-1 transplants. Consequently, the vector-producing MSCs significantly enhanced pro-drug killing of glioma cells compared to MSCs without ability to generate progeny virus. Our study demonstrated the effective MSCs-mediated tumor transduction with progeny vector production to improve suicide gene therapy. Although therapeutic benefit in the various orthotopic or metastatic tumor models has to be further validated, this transduction strategy would eradicate evasive tumors in situ.