Challenges of primate embryonic stem cell research

Embryonic stem (ES) cells hold great promise for treating degenerative diseases, including diabetes, Parkinson's, Alzheimer's, neural degeneration, and cardiomyopathies. This research is controversial to some because producing ES cells requires destroying embryos, which generally means human embryos. However, some of the surplus human embryos available from in vitro fertilization (IVF) clinics may have a high rate of genetic errors and therefore would be unsuitable for ES cell research. Although gross chromosome errors can readily be detected in ES cells, other anomalies such as mitochondrial DNA defects may have gone unrecognized. An insurmountable problem is that there are no human ES cells derived from in vivo-produced embryos to provide normal comparative data. In contrast, some monkey ES cell lines have been produced using in vivo-generated, normal embryos obtained from fertile animals; these can represent a "gold standard" for primate ES cells. In this review, we argue a need for strong research programs using rhesus monkey ES cells, conducted in parallel with studies on human ES and adult stem cells, to derive the maximum information about the biology of normal stem cells and to produce technical protocols for their directed differentiation into safe and functional replacement cells, tissues, and organs. In contrast, ES cell research using only human cell lines is likely to be incomplete, which could hinder research progress, and delay or diminish the effective application of ES cell technology to the treatment of human diseases.     

Embryonic stem cells and cell replacement therapies in the nervous system

Embryonic stem (ES) cells are pluripotential cells derived from the pre-implantation embryo. They can proliferate indefinitely in vitro while retaining pluripotency. ES cells can also be made to differentiate into a large variety of cell types in vitro. This has paved the way to research aimed at using ES-derived cells for cell replacement therapies. Hence, mouse ES cells can efficiently differentiate into neural precursors which can further generate functional neurons, astrocytes, and oligodendrocytes. Methods have also been developed to coax mouse ES-derived neural stem cells to differentiate into either dopaminergic neurons or motoneurons. Mouse ES-derived neural stem cells, or their fully differentiated progeny, have been shown to survive, integrate, and to some extent, function following transplantation within appropriate rodent host tissue. Research on human ES cells is still in its infancy. Considerable work has to be done: (1) to master growth and genetic manipulation of human ES cells; (2) to master their differentiation into specific cell types; and (3) to demonstrate that they can provide long term therapeutical benefits upon grafting into damaged tissues in humans. From the ethical point of view, the establishment of appropriate primate model will be an obligatory prerequisite to clinical trials based on ES cells derivatives grafting.     

Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates

Embryonic stem (ES) cell lines provide an invaluable research tool for genetic engineering, developmental biology and disease models. These cells can be maintained indefinitely in culture and yet maintain competence to produce all the cells within a fetus. While mouse ES cell lines were first established over two decades ago and primate ES cells in the 1990 s, validated ES cell lines have yet to be established in ungulates. Why competent, pluripotent ES cells can be established from certain strains of mice and from primates, and not from cows, sheep, goats or pigs is an on-going topic of interest to animal reproduction scientists. The identification of appropriate stem cell markers, functional cytokine pathways, and key pluripotency-maintaining factors along with the release of more comprehensive bovine and porcine genomes, provide encouragement for establishment of ungulate ES cell lines in the near future.     

胚胎干细胞

胚胎干细胞具有自我复制并分化为人体各种功能细胞的潜能。胚胎干细胞具有的独特生物学特性使其被广泛应用于生物学研究的各个领域,特别是发育学。同时,它潜在的医学应用也成为世界范围内的研究热点。但是,由于人胚胎干细胞的来源为植入前的早期胚胎,人胚胎干细胞自诞生之日起便倍受争议。本文将从胚胎干细胞的来源、特性、鉴定标准、增殖机理、应用前景以及研究本身涉及的伦理学争论给予概述。     

Telomerase inhibition promotes an initial step of cell differentiation of primate embryonic stem cell

Embryonic stem (ES) cell is well known as a totipotent cell, which is derived from a blastcyst and has potential to differentiate into every kind of somatic cell. ES cell bears self-renewal characteristic as well as differentiation potential. ES cell bears telomerase activity to avoid telomere shortening, which is a characteristic of differentiated somatic cells. As the differentiation of ES cells proceeds, their telomerase activity is losing. However, it has not been convinced whether suppression of the telomerase activity promotes progression of ES cell differentiation. The effect of telomerase inhibitor on the differentiation potential of marmoset ES cell was assessed, counting cells expressing embryonic markers (alkaline phosphatase and TPA-1-60) under existence of a telomerase inhibitor. Telomerase inhibitor showed a promotional effect for the marmoset ES cell differentiation. This result suggests that exogenous inhibition of telomerase activity leads to induction of an early differentiation of primate ES cell.     

Self-renewal vs. differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells are typically derived from the inner cell mass of the preimplantation blastocyst and can both self-renew and differentiate into all the cells and tissues of the embryo. Because they are pluripotent, ES cells have been used extensively to analyze gene function in development via gene targeting. The embryonic stem cell is also an unsurpassed starting material to begin to understand a critical, largely inaccessible period of development. If their differentiation could be controlled, they would also be an important source of cells for transplantation to replace cells lost through disease or injury or to replace missing hormones or genes. Traditionally, ES cells have been differentiated in suspension culture as embryoid bodies, named because of their similarity to the early postimplantation-staged embryo. Unlike the pristine organization of the early embryo, differentiation in embryoid bodies appears to be largely unpatterned, although multiple cell types form. It has recently been possible to separate the desired cell types from differentiating ES cells in embryoid bodies by using cell-type-restricted promoters driving expression of either antibiotic resistance genes or fluorophores such as EGFP. In combination with growth factor exposure, highly differentiated cell types have successfully been derived from ES cells. Recent technological advances such as RNA interference to knock down gene expression in ES cells are also producing enriched populations of cells and elucidating gene function in early development.     

Differentiation of rhesus embryonic stem cells to neural progenitors and neurons

Embryonic stem (ES) cells are pluripotent cells capable of differentiating into cell lineages derived from all primary germ layers including neural cells. In this study we describe an efficient method for differentiating rhesus monkey ES cells to neural lineages and the subsequent isolation of an enriched population of Nestin and Musashi positive neural progenitor (NP) cells. Upon differentiation, these cells exhibit electrophysiological characteristics resembling cultured primary neurons. Embryoid bodies (EBs) were formed in ES growth medium supplemented with 50% MEDII. After 7 days in suspension culture, EBs were transferred to adherent culture and either differentiated in serum containing medium or expanded in serum free medium. Immunocytochemistry on differentiating cells derived from EBs revealed large networks of MAP-2 and NF200 positive neurons. DAPI staining showed that the center of the MEDII-treated EBs was filled with rosettes. NPs isolated from adherent EB cultures expanded in serum free medium were passaged and maintained in an undifferentiated state by culture in serum free N2 with 50% MEDII and bFGF. Differentiating neurons derived from NPs fired action potentials in response to depolarizing current injection and expressed functional ionotropic receptors for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). NPs derived in this way could serve as models for cellular replacement therapy in primate models of neurodegenerative disease, a source of neural cells for toxicity and drug testing, and as a model of the developing primate nervous system.     

The origin and identity of embryonic stem cells

Embryonic stem (ES) cells are used extensively in biomedical research and as a model with which to study early mammalian development, but their exact origin has been subject to much debate. They are routinely derived from pre-implantation embryos, but it has been suggested that the cells that give rise to ES cells might arise from epiblast cells that are already predisposed to a primordial germ cell (PGC) fate, which then progress to ES cell status via the PGC lineage. Based on recent findings, we propose here that ES cells can be derived directly from early epiblast cells and that ES cells might arise via two different routes that are dictated by their culture conditions.     

Regulation beyond genome sequences: DNA and histone methylation in embryonic stem cells

Embryonic stem (ES) cells distinct themselves from other cell type populations by their pluripotent ability. The unique features of ES cells are controlled by both genetic and epigenetic factors. Studies have shown that the methylation status of DNA and histones in ES cells is quite different from that of differentiated cells and somatic stem cells. Herein, we summarized recent advances in DNA and histone methylation studies of mammalian ES cells. The methylation status of several key pluripotent regulatory genes is also discussed.     

胚胎干细胞向肝细胞诱导分化

胚胎干细胞具有分化成三胚层细胞的潜能。它已被视为治疗多种疾痛的一种新兴策略。在现阶段,通过不同的诱导途径可将胚胎干细胞诱导成为肝细胞：体外诱导、体内诱导以及体外和体内相结合诱导分化。然而从体内实验结果来看,其嵌合率及分化率不高,这是一个亟需解决的问题,否则就无法成功地将其应用于临床治疗。     

锌指核酸内切酶:基因操作的有力工具

针对动植物进行基因靶向操作的技术,是解析基因功能、研究疾病,以及农业经济生产中一个有用的工具。至今,基因靶向操作主要是通过在胚胎干细胞(embryonic stem cell,ES cell)中进行同源重组或者是体细胞核转移的方法进行,但同源重组方法由于需要ES细胞而被限制在个别物种,而核转移方法存在核去分化、效率低、成本高的缺陷。近几年,一种基于锌指核酸内切酶(zinc-finger nuclease,ZFN)基因靶向修饰的新技术被应用于包括植物、果蝇、爪蟾、斑马鱼和大鼠等不同物种的基因操作。通过胚胎注射ZFN的质粒或是mRNA可以有效地定靶并迅速地在内源基因上引起可遗传的突变。ZFN介导基因靶向敲除的可行性,使得那些无法获得ES细胞和克隆技术支持的物种的基因靶向修饰成为可能。     

Generation of skeletal muscle from transplanted embryonic stem cells in dystrophic mice

Embryonic stem (ES) cells have great therapeutic potential because of their capacity to proliferate extensively and to form any fully differentiated cell of the body, including skeletal muscle cells. Successful generation of skeletal muscle in vivo, however, requires selective induction of the skeletal muscle lineage in cultures of ES cells and following transplantation, integration of appropriately differentiated skeletal muscle cells with recipient muscle. Duchenne muscular dystrophy (DMD), a severe progressive muscle wasting disease due to a mutation in the dystrophin gene and the mdx mouse, an animal model for DMD, are characterized by the absence of the muscle membrane associated protein, dystrophin. Here, we show that co-culturing mouse ES cells with a preparation from mouse muscle enriched for myogenic stem and precursor cells, followed by injection into mdx mice, results occasionally in the formation of normal, vascularized skeletal muscle derived from the transplanted ES cells. Study of this phenomenon should provide valuable insights into skeletal muscle development in vivo from transplanted ES cells.     

In vitro differentiation of murine embryonic stem cells into keratinocyte-like cells

Embryonic stem (ES) cells are omnipotent; they can differentiate into every cell type of the body. The development of culture conditions that allow their differentiation has made it conceivable to produce large numbers of cells with lineage-specific characteristics in vitro. Here, we describe a method by which murine ES cells can be differentiated into cells with characteristics of epidermal keratinocytes. Keratinocyte-like cells were isolated from embryoid bodies and grown in culture. Potential applications of this method are the in vitro differentiation of cells of interest from ES cells of mice with lethal phenotypes during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments.     

胚胎干细胞生物学特性及其应用前景

胚胎干细胞是来源于着床前的囊胚内细胞团或早期胎儿的原始生殖细胞的一类未分化的全能性（多能性）干细胞,具有无限增殖和全能化的潜力,胚胎干细胞在发育生物学基础研究,动物胚胎工程研究生产和临床医学上具有诱人的应用前景。     

维持胚胎干细胞不分化状态的分子机制

胚胎干细胞（embryonic stem cell,ESC）是指从早期胚胎的囊胚内细胞团（inner cell mass,ICM）分离出来的具有自我更新和多向分化潜能的细胞,目前被广泛地应用于基础研究和临床应用研究等生命科学领域。ESC在体外培养过程中维持不分化状态是其应用的前提与基础,阐明这个分子机制非常必要。文章总结了维持hESC未分化状态机制的最新进展,主要介绍在维持ESC不分化过程中,分化抑制因子LIF、Oct-3/4及Nanog等的重要作用。