In vitro enhanced differentiation of neural networks in ES gut-like organ from mouse ES cells by a 5-HT4-receptor activation

Using an embryoid body (EB) culture system, we developed a functional organ-like cluster, a “gut”, from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited various types of spontaneous movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (ICC) (c-kit, a transmembrane receptor that has tyrosine kinase activity, positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified, but enteric neural networks were not identified. In the present study, we succeeded in forming dense enteric neural networks by a 5-HT₄-receptor (SR4) agonist, mosapride citrate (1–10 μM) added only during EB formation. Addition of an SR4-antagonist, GR113808 (10 μM) abolished the SR4-agonist-induced formation of enteric neural networks. The SR4-agonist (1 μM) up-regulated the expression of mRNA of SR4 and the SR4-antagonist abolished this upregulation. 5-HT per se exerted similar effects to those of SR4-agonist, though less potent. These results suggest SR4-agonist differentiated enteric neural networks, mediated via activation of SR4 in the ES gut.     

The first reported generation of human induced pluripotent stem cells (iPS cells) and iPS cell-derived cardiomyocytes in the Netherlands

One of the recent breakthroughs in stem cell research has been the reprogramming of human somatic cells to an embryonic stem cell (ESC)-like state (induced pluripotent stem cells, iPS cells). Similar to ESCs, iPS cells can differentiate into derivatives of the three germ layers, for example cardiomyocytes, pancreatic cells or neurons. This technique offers a new approach to investigating disease pathogenesis and to the development of novel therapies. It may now be possible to generate iPS cells from somatic cells of patients who suffer from vascular genetic diseases, such as hereditary haemorrhagic telangiectasia (HHT). The iPS cells will have a similar genotype to that of the patient and can be differentiated in vitro into the cell type(s) that are affected in the patient. Thus they will serve as excellent models for a better understanding of mechanisms underlying the disease. This, together with the ability to test new drugs, could potentially lead to novel therapeutic concepts in the near future. Here we report the first derivation of three human iPS cell lines from two healthy individuals and one HHT patient in the Netherlands. The iPS cells resembled ESCs in morphology and expressed typical ESC markers. In vitro, iPS cells could be differentiated into cells of the three germ layers, including beating cardiomyocytes and vascular cells. With this technique it will be possible to establish human cardiovascular disease models from patient biopsies provided by the principal hospitals in the Netherlands. (Neth Heart J 2010;18:51-4.)     

诱导多能干细胞在心血管疾病研究和治疗中的应用

诱导多能干细胞（induced pluripotent stem cells,iPS细胞）不仅具有与胚胎干细胞（embryonic stem cell,ESC）相似的各项特性,相对于ESC,iPS细胞,尤其患者特异性iPS细胞还具有来源方便、不存在免疫排斥和伦理问题以及可以保留特定个体基因型等优点,为再生医学提供了可能的细胞来源。该文主要从心血管药物的筛选、疾病模型的建立、iPS细胞应用于心脏移植研究等方面入手,探讨了iPS细胞在心血管疾病研究和治疗中的现状和未来。     

诱导多能干细胞研究进展

人类诱导多能干细胞（induced pluripotent stem cells,iPS细胞）的建立被公认为目前最重要的科技进展之一。iPS细胞在动物疾病模型上的成功治疗,病患特异性iPS细胞的研究及iPS细胞的定向分化研究将有可能使人们避开治疗性克隆的伦理和技术障碍,给人类疾病的干细胞治疗带来光明的前景。本文从iPS细胞的诱导策略和方法,来源细胞及筛选、重编程机制的研究现状、应用前景以及研究中存在的问题等方面对其作一综述和讨论。     

iPS细胞研究的新进展及应用

通过导入特定的转录因子可将分化的体细胞重编程为诱导性多能干细胞(Induced pluripotent stem cells,iPS cells),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,是生命科学领域的一次巨大革命。与胚胎干细胞(Embryonic stem cells,ES cells)一样,iPS细胞能够自我更新并维持未分化状态,在体内可分化为3个胚层来源的所有细胞,进而参与形成机体所有组织和器官。在体外,iPS细胞可定向诱导分化出多种成熟细胞。因此,iPS细胞在理论研究和临床应用等方面都极具应用价值。文章对iPS细胞诱导的最新研究进展、iPS细胞诱导的不同方法,如何提高iPS细胞的制备效率和安全性,iPS细胞在基础研究以及临床研究等方面的应用进行了全面综述,并探讨了iPS细胞研究领域面临的问题以及该技术在转基因动物研究中的发展前景。     

Induced pluripotent stem cells: a new technology to study human diseases

Induced pluripotent stem cells (iPS cells) are somatic cells that have been reprogrammed to a pluripotent state by the introduction of specific factors. They can be generated from cells of different origins such as fibroblasts, keratinocytes, hepatocytes and blood. iPS cells are similar to embryonic stem cells in several aspects such as morphology, expression of pluripotency markers and the capacity to develop teratomas; tumors containing cells of the three germ layers. As pluripotent stem cells they can be differentiated into several lineages including neuronal, cardiac and blood cells. Recently, several groups have successfully generated patient-specific iPS cells from donors suffering different disorders and differentiated them into the cell type affected by the disease. These new human cell-based models cannot only be used to study the dynamics of diseases but also as systems to screen new drugs. Moreover, iPS cells promise to be good candidates for regenerative medicine.     

c-Myc对诱导性多能干细胞的诱导效率及全能性影响的研究

该研究探讨转录因子c—Myc对多能诱导干细胞（inducedpluripotentstemcells,iPS）诱导效率及形成的iPS克隆全能性的影响。将Yam,dnaka四因子（Oct4、Sox2、Klf4和C—Myc,OSKM）和不舍C。Myc的三因子（OSK）病毒分别感染OG2小鼠成纤维细胞（mouseembryofibroblast,MEF）,诱导成为iPSN胞,通过计数iPS克隆形成数目和流式细胞仪分析iPS克隆的绿色荧光蛋白（GFP）阳性比例。比较OSKM和OSK诱导iPS的效率。分别挑取三株OSK和OSKM诱导的iPS克隆,采用荧光定量PCR法、碱性磷酸酶fAP）染色法和免疫荧光法检测iPS克隆的干性相关蛋白的表达;采用畸胎瘤实验、嵌合体形成实验和四倍体补偿实验检测iPS克隆的全能性。结果显示：在该实验室诱导体系里,OSK病毒感染MEF后形成的iPS克隆数目明显增多,GFP阳性克隆的比例增加。OSK和OSKM诱导iPS克隆的干细胞基本特征,包括形态、AP染色、干细胞特异性基因表达、三胚层形成均没有明显差异。然而,OSK诱导iPS克隆在形成嵌合体小鼠的全能性评估实验中的阳性比例高于OSKM克隆,且只有OSK克隆株能够获得生殖系传递小鼠和四倍体补偿小鼠。该研究表明,在实验诱导体系里去除C—Myc的三因子OSK诱导iPS的效率显著高于OSKM四因子,且三因子诱导iPS克隆具有更好的全能性。     

诱导性多潜能干细胞(iPS cells)——现状及前景展望

主要从 iPS细胞发展历程、获得 iPS细胞的几个关键步骤 (如基因导入方式、诱导 iPS细胞所需因子组合与小分子化合物运用和体细胞种类选择等)、病人或疾病特异性 iPS细胞、iPS细胞体内外诱导分化与其衍生物的临床应用和制备无遗传修饰的(genetic modification-free) iPS细胞的可行性与前景等方面对 iPS细胞最新研究进展做评述．日本和美国研究小组先后用4种基因将小鼠(2006年8月)和人(2007年11～12月)的体细胞在体外重编程为诱导性多潜能干细胞(induced pluripotent stem cells,iPS cells),此后在短短两年多时间内,iPS 细胞的研究和关注度呈爆炸式增长．体细胞重编程、去分化和多潜能干细胞来源等一系列热点问题再次成为干细胞和发育生物学等研究的热点和焦点．与胚胎干细胞(embryonic stem cells,ES cells)一样,iPS细胞在体内可分化为3个胚层来源的所有细胞,进而参与形成机体所有组织和器官．迄今,在体外已由 iPS细胞定向诱导分化出功能性的多种成熟细胞．因此,iPS细胞研究不仅具有重要理论意义,而且在再生医学、组织工程和药物发现与评价等方面极具应用价值．     

Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells

Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and after differentiation. Analysed cell lines were derived soon after PGC specification, “early”, in comparison with EG cells derived after PGC colonisation of the genital ridge, “late” and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell and ES cell lines. We also observed that all analysed XX cell lines exhibited less methylation than XY. We suggest that this heterogeneity may reflect the changes in DNA methylation taking place in the germ cell lineage soon after specification.     

核移植与诱导多能干细胞技术在体细胞重编程研究中的应用

体细胞重编程是指分化的体细胞在特定的条件下被逆转后恢复到多能性或全能性状态,或者形成多能干细胞系,或者形成早期胚胎然后发育成一个新的个体的过程。诱导体细胞重编程的方法有许多,如核移植（nuclear transfer,NT）、细胞融合、细胞培养和通过导入特定因子获得诱导多能干（induced pluripotent stem,iPS）细胞的方法等。其中核移植和iPS技术是到目前为止诱导体细胞为多能干细胞最为完全、最具有运用于临床再生医学潜能的方法。然而,它们的效率都很低,机制也不清楚,如何将两个方法结合在一起,提高重编程的效率,揭示重编程的机制,进而促进其在患者特异性治疗中的运用将是下阶段的努力方向。     

Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate

During embryonic development, organs arise along the gut tube as a series of buds in a stereotyped anterior-posterior (A-P) pattern. Using chick-quail chimeras and in vitro tissue recombination, we studied the interactions governing the induction and maintenance of endodermal organ identify focusing on the pancreas. Though several permissive signals in pancreatic development have been previously identified, here we provide evidence that lateral plate mesoderm sends instructive signals to the endoderm, signals that induce expression of the pancreatic genes Pdx1, p48, Nkx6.1, glucagon, and insulin. Moreover, this instructive signal directs cells to form ectopic insulin-positive islet-like clusters in endoderm that would otherwise form more rostral organs. Once generated, endocrine cells no longer require interaction with mesoderm, but nonendocrine cells continue to require permissive signals from the mesoderm. Stimulation of activin, BMP, or retinoic acid signaling is sufficient to induce Pdx1 expression in endoderm anterior to the pancreas. Lateral plate mesoderm appears to pattern the endoderm in a posterior-dominant fashion as first noted in the patterning of the neural tube at the same embryonic stage. These findings argue for a central role of the mesoderm in coordinating the A-P pattern of all three primary germ layers.     

利用piggyBac转座子制备牛成体多能干细胞诱导技术研究

通过逆转录病毒等媒介表达核转录因子Oct4、Sox2、Klf4、c-Myc可将体细胞重编程为诱导多能干细胞(induced pluripotent stem cells, iPSc)。时至今日,已经报道了小鼠、人、大鼠、猪、羊、马、牛的iPS细胞,但大动物iPS的多能性特别是嵌合体形成和生殖细胞传代还没有得到确认。与逆转录病毒等不同的是,piggyBac转座子转染效率高且无病毒源性、操作简单,可以在转座酶的存在下被安全切除。首次尝试了采用piggyBac转座子携带鼠源Oct4、Sox2、Klf4、c-Myc、Rarg和Lrh16个核转录因子诱导胎牛成纤维细胞,成功获得牛类iPS细胞,其形态与小鼠胚胎干细胞相似,克隆边界清晰、呈丘状、克隆内细胞致密、核大。RT-PCR与免疫组织化学染色分析均显示牛类iPS细胞表达多能性基因。该类细胞体外诱导分化可形成类胚体EB,且表达3个胚层的基因;体内诱导分化可形成畸胎瘤,苏木精、伊红染色显示瘤体有三胚层的分化。上述结果显示利用piggyBac转座子制备牛多潜能干细胞诱导技术可行,产生的牛类iPS细胞具有潜在多能性。     

How induced pluripotent stem cells are redefining personalized medicine

Since the generation of the first induced pluripotent stem (iPS) cells, the stem cell field has grown at an unparalleled pace. Today, these cells have become the major tools in the advancement of personalized medicine. Here we review the experiments that lead to their discovery as well as the latest developments in iPS cell biology. By emphasizing the current applications and limitations of induced pluripotency, we discuss how iPS cells are shaping innovation in personalized therapies. In addition, we analyze the major landmarks in direct lineage reprogramming, a potentially faster alternative to the use of iPS cells in therapy. Finally, we present the current progress in disease modeling and future directions of the treatment of genetic disorders.     

Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro

Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm. We established differentiation protocols for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiomyocytes, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells. During differentiation, tissue-specific genes, proteins, ion channels, receptors and action potentials were expressed in a developmentally controlled pattern. This pattern closely recapitulates the developmental pattern during embryogenesis in the living organism. In vitro, the controlled developmental pattern was found to be influenced by differentiation and growth factor molecules or by xenobiotics. Furthermore, the differentiation system has been used for genetic analyses by ‘gain of function’ and ‘loss of function’ approaches in vitro. This revised version was published online in July 2006 with corrections to the Cover Date.     

Directed differentiation of induced pluripotent stem cells towards T lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies (1). CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines (2-7). However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies. TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases (8-10). However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic (11-13), HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture (14-16). Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs. Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.