Organotypic heart slices for cell transplantation and physiological studies

Recent studies have significantly improved our ability to investigate cell transplantation and study the physiology of transplanted cells in cardiac tissue. Several previous studies have shown that fully-immersed heart slices can be used for electrophysiological investigations. Additionally, ischemic heart slices induced by glucose and oxygen deprivation offer a useful tool to investigate mechanical integration and to measure forces of contraction of engrafted cells, at least for short term analysis. A recent and novel model of heart slices, prepared from rat and human tissues, can be maintained in culture for up to two months. This new heart slice model can be used for long term in vitro cell transplantation studies and for pharmacological evaluation. This review will focus on describing these models and demonstrating the use of organotypic heart slices as a novel tool for drug, for studying electrophysiology and for developing cellular therapeutic approaches to alleviate cardiac tissue damage.     

Organotypic heart slices for cell transplantation and physiological studies

Recent studies have significantly improved our ability to investigate cell transplantation and study the physiology of transplanted cells in cardiac tissue. Several previous studies have shown that fully-immersed heart slices can be used for electrophysiological investigations. Additionally, ischemic heart slices induced by glucose and oxygen deprivation offer a useful tool to investigate mechanical integration and to measure forces of contraction of engrafted cells, at least for short term analysis. A recent and novel model of heart slices, prepared from rat and human tissues, can be maintained in culture for up to two months. This new heart slice model can be used for long term in vitro cell transplantation studies and for pharmacological evaluation. This review will focus on describing these models and demonstrating the use of organotypic heart slices as a novel tool for drugs for studying electrophysiology and developing cellular therapeutic approaches to alleviate cardiac tissue damage.Key words: heart, organotypic, culture, stem cells, transplantation, electrophysiology, pharmacology     

Transgenic farm animals - A critical analysis

The notion of directly introducing new genes or otherwise manipulating the genotype of an animal is conceptually straightforward and appealing from the standpoints of both speed and precision with which phenotypic changes can be made. Thus, it is little wonder that the imagination of many animal scientists has been captivated by the success others have achieved in introducing foreign genes into mice. Transgenic mice not only exhibit unique phenotypes, but they also pass those traits on to their progeny. However, before transgenic farm animals become a common component of the livestock industry, a number of formidable obstacles must be overcome. In this review we attempt to identify the critical issues that should be considered by both those currently working in the field and those scientists considering the feasibility of initiating a transgenic livestock project. The inefficiency of producing transgenic animals has been well documented. This does not constrain investigators using laboratory animal models, but it has a major impact on applying transgenic technology to farm animals. The molecular mechanisms of transgene integration have not been elucidated, and as a consequence it is difficult to design strategies to improve the efficiency of the process. In addition to the problems associated with integration of new genes, there are inefficiencies associated with collecting and culturing fertilized eggs as well as embryo transfer in farm animals. Transgenic farm animal studies are major logistical undertakings. Even in the face of these practical hindrances, some may be pressured by administrators to embrace this new technology. As powerful as the transgenic animal model system is, currently there are limits to the kinds of agricultural questions that can be addressed. Some uses are so appealing, however, that several commercial organizations have explored this technology. Within the next decade or two, it is likely that many of the technical hurdles will be overcome. Combining new techniques with a better understanding of the genetic control of physiological systems will make it possible to improve the characteristics of farm animals in highly imaginative ways.     

影响动物细胞同源重组发生与基因打靶效率的分子机制

真核细胞的基因打靶是基因结构与功能研究的一种非常有价值的技术,也是可应用于基因治疗的具有潜力的工具。有2个限制因素束缚真核细胞基因打靶的发展,即同源重组(HR)率非常低而随机整合率非常高。通过特定基因的过表达或表达干涉,使一些参与DNA重组的蛋白表达水平瞬间改变,可能会增加HR率,降低随机整合率。本文列举了一些与HR相关的候选基因,详细介绍了其中的Rad52上位簇基因,还讨论了打靶载体的设计与修饰、DNA转染方法的有效性等。     

Gene expression profiles in the diagnosis and prognosis of cancer

DNA-microarray technology can be used to assess the expression of several thousands of genes at the same time. The identification of the gene expression profiles may help to better characterize human cancer. These studies may reveal subclasses of tumor types with similar histopathologic profile but different clinical courses.Furthermore,such studies could help to define therapeutic sensitivity and to estimate prognosis of various cancers. Identification of gene expression profiles of cancer can identify new therapeutic targets or cancer susceptibility genes. The DNA-microarray technology may write a new chapter in molecular oncology.     

Evolutionary Novelties: How Fish Have Built a Heater Out of Muscle

The evolution of any complex morphology or physiological adaptationinvolves the historical transformation of numerous interactingcomponents from an ancestral to a derived state. How such transformationsoccur are central to our understanding of how novel morphologiesarise. The rapid explosion of technology in the field of molecularbiology provides new tools that can be incorporated into studiesexamining the origin of novel phenotypes. Molecular biologicaltechniques can now be used to probe how changes in gene expressionresult in pathways leading to novel or altered morphologies.The integration of molecular approaches into problems in organismalbiology provides a promising new direction for the analysisof form and function. Interdisciplinary studies, combining theresolving power of molecular biologywith the complex problemsof organismal biology, will shed new light on whole animal functionand evolution.     

Trafficking and cellular distribution of voltage-gated sodium channels

Electrical excitability in cells such as neurons and myocytes depends not only upon the expression of voltage-gated sodium channels but also on their correct targeting within the plasma membrane. Placing sodium channels within a broader cell biological context is beginning to shed new light on a variety of important questions such as the integration of neuronal signaling. Mutations that affect sodium channel trafficking have been shown to underlie several life-threatening conditions including cardiac arrhythmias, revealing an important clinical context to these studies.     

Translational aspects of cardiac cell therapy

Cell therapy has been intensely studied for over a decade as a potential treatment for ischaemic heart disease. While initial trials using skeletal myoblasts, bone marrow cells and peripheral blood stem cells showed promise in improving cardiac function, benefits were found to be short‐lived likely related to limited survival and engraftment of the delivered cells. The discovery of putative cardiac ‘progenitor’ cells as well as the creation of induced pluripotent stem cells has led to the delivery of cells potentially capable of electromechanical integration into existing tissue. An alternative strategy involving either direct reprogramming of endogenous cardiac fibroblasts or stimulation of resident cardiomyocytes to regenerate new myocytes can potentially overcome the limitations of exogenous cell delivery. Complimentary approaches utilizing combination cell therapy and bioengineering techniques may be necessary to provide the proper milieu for clinically significant regeneration. Clinical trials employing bone marrow cells, mesenchymal stem cells and cardiac progenitor cells have demonstrated safety of catheter based cell delivery, with suggestion of limited improvement in ventricular function and reduction in infarct size. Ongoing trials are investigating potential benefits to outcome such as morbidity and mortality. These and future trials will clarify the optimal cell types and delivery conditions for therapeutic effect.     

Medical simulation: the new tool for training and skill assessment

Medical simulation is a new method to facilitate skill training and assessment. Simulation has achieved a high degree of sophistication in aviation and other fields. However, the complexity of health care, the numerous stakeholders, and the lack of central control of medical education have been barriers to the development and broad implementation of medical simulation. Acceptance by the medical community is growing, with the publication of scientific validation studies, the development of economic models and funding, and the integration of simulation into existing curricula and training programs. The major forces for implementing simulation will most likely come from the medical device industry and from institutions with mandates to improve the quality of health care and enhance patient safety. Certification boards are expected to increase their utilization of simulation technology to objectively assess proficiency of skills relevant to physicians and the health care system. Medical simulation has made the transition from an experimental technology to the clinical world, and the next five to 10 years may be viewed as the golden age of medical simulation.     

Site-specific gene integration technologies for crop improvement

Targeted integration of foreign genes into plant genomes is a much sought-after technology for engineering precise integration structures. Homologous recombination-mediated targeted integration into native genomic sites remained somewhat elusive until made possible by zinc finger nuclease-mediated double-stranded breaks. In the meantime, an alternative approach based on the use of site-specific recombination systems has been developed which enables integration into previously engineered genomic sites (site-specific integration). Follow-up studies have validated the efficacy of the site-specific integration technology in generating transgenic events with a predictable range and stability of expression through successive generations, which are critical features of reliable and practically useful transgenic lines. Any DNA delivery methods can be used for site-specific integration; however, best efficiency is mostly obtained with direct DNA delivery methods such as particle bombardment. Although site-specific integration approach provides unique advantages for producing transgenic plants, it is still not a commonly used method. The present article discusses barriers and solutions for making it readily available to both academic research and applicative use.     

Rarity of tagSNPs across transferred mtDNA inserts in human genome

The study deals with the single nucleotide polymorphism (SNPs, HapMap data) around the mtDNA insertions in human genome. The results obtained from this study suggest that application of tagSNP approach for large scale genotyping targeting NUMT integration sites may be difficult due to lack of informative mutations around these loci. This warrants development of new approaches to tag mtDNA inserts in genome-wide association studies.     

Rarity of TagSNPs across transferred mtDNA inserts in human genome

The study deals with the single nucleotide polymorphism (SNPs, HapMap data) around the mtDNA insertions in human genome. The results obtained from this study suggest that application of tagSNP approach for large scale genotyping targeting NUMT integration sites may be difficult due to lack of informative mutations around these loci. This warrants development of new approaches to tag mtDNA inserts in genome-wide association studies.     

Cardiac matrix remodeling following intracoronary cell transplantation in dilated cardiomyopathic rabbits

Cellular cardiomyoplasty has been proposed as a promising therapeutic strategy for chronic heart failure. Previous studies focused on structural changes in cardiomyocytes to explain the potential benefits for contractile function. However, limited information is available about the cardiac matrix remodeling following cell transplantation in dilated cardiomyopathy (DCM). Here, we established a new animal model of intracoronary bone marrow mononuclear cells (BMMNCs) transplantation to explore extracellular matrix remodeling in adriamycin-induced cardiomyopathic rabbits. In vivo studies demonstrated that BMMNCs transplantation can dramatically delay the progress of collagen metabolism and decrease myocardial collagen volume fraction. The beneficial effects were mediated by attenuating stress-generated over-expression of matrix metalloproteinases (MMPs) in ventricular remodeling. Improved cardiac function may be contributed in part by stem-associated inhibition of extracellular matrix remodeling.     

Mesenchymal stem cells and the treatment of cardiac disease

The ischemia-induced death of cardiomyocytes results in scar formation and reduced contractility of the ventricle. Several preclinical and clinical studies have supported the notion that cell therapy may be used for cardiac regeneration. Most attempts for cardiomyoplasty have considered the bone marrow as the source of the "repair stem cell(s)," assuming that the hematopoietic stem cell can do the work. However, bone marrow is also the residence of other progenitor cells, including mesenchymal stem cells (MSCs). Since 1995 it has been known that under in vitro conditions, MSCs differentiate into cells exhibiting features of cardiomyocytes. This pioneer work was followed by many preclinical studies that revealed that ex vivo expanded, bone marrow-derived MSCs may represent another option for cardiac regeneration. In this work, we review evidence and new prospects that support the use of MSCs in cardiomyoplasty.     

Sweet,yet underappreciated: Proteoglycans and extracellular matrix remodeling in heart disease

Extracellular matrix remodeling is extensive in several heart diseases and hampers cardiac filling, often leading to heart failure. Proteoglycans have over the last two decades emerged as molecules with important roles in matrix remodeling and fibrosis in the heart. Here we discuss and review current literature on proteoglycans that have been studied in cardiac remodeling. The small leucine rich proteoglycans (SLRPs) are located within the extracellular matrix and are organizers of the matrix structure. Membrane-bound proteoglycans, such as syndecans and glypicans, act as receptors and direct cardiac fibroblast signaling. Recent studies indicate that proteoglycans are promising as diagnostic biomarkers for cardiac fibrosis, and that they may provide new therapeutic strategies for cardiac disease.     

Electrostimulation induces cardiomyocyte predifferentiation of fibroblasts

Stem-cell therapy has become a promising therapeutic tool for myocardial repair. Cardiac pre-committed cells, which complete their differentiation in the myocardium, may reduce fibrosis and restore muscle function. However, many questions concerning a precise, functional integration of injected cells remain unanswered. Fibroblasts regulate the cardiac extracellular matrix and are the most abundant cell population in an infarcted area. Electrostimulation is a well-known trophic factor and can induce phenotypic changes in myoblasts. The objective of this study was to evaluate the effectiveness of electrical stimulation to induce pre-commitment of fibroblasts into cardiomyocytes in vitro. Using short-time electrostimulation in a cytokine-free culture system, we induced pre-commitment of two fibroblast cell lines to a cardiomyocyte phenotype. This partial differentiation in vitro may facilitate further differentiation within the cardiac environment and result in better electro-mechanical integration of the therapeutically introduced cells.     

T-DNA转移及整合的分子机制研究进展

农杆菌介导的外源基因转化是目前植物转基因应用比较广泛的方法。近年来关于农杆菌介导的整合机制的研究已经取得了很大的进步。研究表明,在VirD2和VirE2协助下,农杆菌转移T-DNA进入植物细胞,这两种蛋白共同协助T-DNA完成转移、核定位及在植物基因组中的整合。近年来关于拟南芥突变体的研究表明,被转化植物的宿主基因在T-DNA转移及整合过程中发挥主要的作用。通过对现有研究成果详细讨论了Vir’蛋白(VirD2和VirE2)及植物基因在农杆菌介导植物转化中的作用,详细讨论了依靠VirD2蛋白和SDSA(synthesis-dependent strand-annealing)整合的两类不同的整合模式,根据最新的研究成果阐述了以基因组的双链断裂修复为基础的整合模型,并提出新的观点。     

Single-Batch Production of Recombinant Human Polyclonal Antibodies

We have previously described the development and implementation of a strategy for production of recombinant polyclonal antibodies (rpAb) in single batches employing CHO cells generated by site-specific integration, the Sympress^TM I technology. The Sympress^TM I technology is implemented at industrial scale, supporting a phase II clinical development program. Production of recombinant proteins by site-specific integration, which is based on incorporation of a single copy of the gene of interest, makes the Sympress^TM I technology best suited to support niche indications. To improve titers while maintaining a cost-efficient, highly reproducible single-batch manufacturing mode, we have evaluated a number of different approaches. The most successful results were obtained using random integration in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. This new expression process is termed the Sympress^TM II technology. Here we describe proof-of-principle data demonstrating the feasibility of the Sympress^TM II technology for single-batch rpAb manufacturing using two model systems each composed of six target-specific antibodies. The compositional stability and the batch-to-batch reproducibility of rpAb produced by the ECHO cells were at least as good as observed previously using site-specific integration technology. Furthermore, the new process had a significant titer increase.