Size isn''t everything: lessons in genetic miniaturisation from nucleomorphs

Nucleomorphs are the vestigial nuclear genomes of eukaryotic algal cells now existing as endosymbionts within a host cell. Molecular investigation of the endosymbiont genomes has allowed important insights into the process of eukaryote/eukaryote cell endosymbiosis and has also disclosed a plethora of interesting genetic phenomena. Although nucleomorph genomes retain classic eukaryotic traits such as linear chromosomes, telomeres, and introns, they are highly reduced and modified. Nucleomorph chromosomes are extremely small and encode compacted genes which are disrupted by the tiniest spliceosomal introns found in any eukaryote. Mechanisms of gene expression within nucleomorphs have apparently accommodated increasingly parsimonious DNA usage by permitting genes to become co-transcribed or, in select cases, to overlap.     

Zebrafish: tools for investigating cellular differentiation

Two recent large-scale genetic screens in zebrafish have identified many mutations that affect differentiation in a variety of organ systems, particularly the notochord, the neural crest and the blood. The combination of these newly identified mutations and well established embryological methods makes zebrafish uniquely suited among vertebrate experimental systems to simultaneously address the roles of specific genes and specific cell—cell interactions during differentiation.     

The self-renewing mechanism of stem cells in the germline

Germline stem cells (GSCs) are a self-renewing population of germ cells that serve as the source of gametes in diverse organisms. Current research suggests that the self-renewing division of GSCs is controlled both by somatic signaling and by intracellular mechanisms such as differential gene expression, asymmetric cytoskeletal organization, and the cell cycle machinery. These findings provide a framework for the further study of GSCs and stem cell renewal in general.     

The Janus face of pluripotent stem cells--connection between pluripotency and tumourigenicity

Pluripotent stem cells have gained special attraction because of their almost unlimited proliferation and differentiation capacity in vitro. These properties substantiate the potential of pluripotent stem cells in basic research and regenerative medicine. Here three types of in vitro cultured pluripotent stem cells (embryonic carcinoma, embryonic stem and induced pluripotent stem cells) are compared in their historical context with respect to their different origin and properties. It became evident that tumourigenicity is an inherent property of pluripotent cells based on p53 down-regulation, expression of tumour-related genes and high telomerase activity that allow unlimited proliferation. In addition, culture-adapted genetic and epigenetic changes may induce tumourigenicity of pluripotent cells. The use of stem cells in regenerative medicine, however, requires non-malignant cell types and strategies that circumvent stages of malignancy.Reprogramming strategies of adult somatic cells that avoid the tumourigenic state of pluripotency may offer alternatives for future biomedical application.     

Safeguarding clinical translation of pluripotent stem cells with suicide genes

The generation of human induced pluripotent stem cells (hiPSCs) opens a new avenue in regenerative medicine. However, transplantation of hiPSC-derived cells carries a risk of tumor formation by residual pluripotent stem cells. Numerous adaptive strategies have been developed to prevent or minimize adverse events and control the in vivo behavior of transplanted stem cells and their progeny. Among them, the application of suicide gene modifications, which is conceptually similar to cancer gene therapy, is considered an ideal means to control wayward stem cell progeny in vivo. In this review, the choices of vectors, promoters, and genes for use in suicide gene approaches for improving the safety of hiPSCs-based cell therapy are introduced and possible new strategies for improvements are discussed. Safety-enhancing strategies that can selectively ablate undifferentiated cells without inducing virus infection or insertional mutations may greatly aid in translating human pluripotent stem cells into cell therapies in the future.     

On growth and form: control of cell morphogenesis in fission yeast

In the past year, we have gained considerable insight into the process of cell morphogenesis and the establishment of positional information in fission yeast. The highlights include a better understanding of the role of the microtubule cytoskeleton in the control of cell shape, as well as the identification of novel genes essential for the establishment of cell polarity and for the positioning of the site of cell division.     

Soluble growth factors stimulate spermatogonial stem cell divisions that maintain a stem cell pool and produce progenitors in vitro

Spermatogonial stem cells (SSCs) support life-long spermatogenesis by self-renewing and producing spermatogonia committed to differentiation. In vitro, SSCs form three-dimensional spermatogonial aggregates (clusters) when cultured with glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2); serial passaging of clusters results in long-term SSC maintenance and expansion. However, the role of these growth factors in controlling patterns of SSC division and fate decision has not been understood thoroughly. We report here that in a short-term culture, GDNF and FGF2 increase the number of dividing SSCs, but not the total SSC number, compared to a no-growth-factor condition. Since the total germ cell number increases with growth factors, these results suggest that GDNF and FGF2 promote a SSC division pattern that sustains the size of the stem cell pool while generating committed progenitors. Our data also show that SSC numbers increase when the cluster structure is disintegrated and cell–cell interaction in clusters is disrupted. Collectively, these results suggest that in this culture system, GDNF and FGF2 stimulate SSC divisions that promote self-renewal and differentiation in the SSC population, and imply that the destruction of the cluster structure, a potential in vitro niche, may contribute to SSC expansion.     

Spermatogonial stem cells

The mammalian seminiferous epithelium consists of a highly complex yet well-organized cell population, with germ cells in mitosis and meiosis and postmeiotic cells undergoing transformation to become spermatozoa. To study the factors which control renewal and differentiation of spermatogonial stem cells, animal models are now available which allow for arrest and restart of spermatogonial differentiation. In addition, marked progress has been made in understanding the control of apoptosis and its role in spermatogonia. For the future, spermatogonial stem cell transplantation may have important practical applications.     

Skeletal muscle stem and progenitor cells: reconciling genetics and lineage

Skeletal muscle provides a unique paradigm for studying stem to differentiated cell transitions, as well as the acquisition of cellular identity. Embryological and genetic studies over the last decades have unveiled key signaling pathways and regulatory genes which are involved in this process. In the adult, regeneration from fiber-associated satellite cells as well as non-muscle cells have opened the perspective for cell therapy studies. Paradoxically, however, the lineage has remained largely elusive. Recent studies have provided clues regarding the cellular organization in this lineage. Furthermore, the complexity of the genetic networks regulating global and local myogenic programs can be correlated with location and lineage. Finally, prenatal and postnatal developmental strategies have similarities and differences which will also be highlighted.     

Neural progenitor genes. Germinal zone expression and analysis of genetic overlap in stem cell populations

The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.     

Immunological considerations for embryonic and induced pluripotent stem cell banking

Recent advances in stem cell technology have generated enthusiasm for their potential to study and treat a diverse range of human disease. Pluripotent human stem cells for therapeutic use may, in principle, be obtained from two sources: embryonic stem cells (hESCs), which are capable of extensive self-renewal and expansion and have the potential to differentiate into any somatic tissue, and induced pluripotent stem cells (iPSCs), which are derived from differentiated tissue such as adult skin fibroblasts and appear to have the same properties and potential, but their generation is not dependent upon a source of embryos. The likelihood that clinical transplantation of hESC- or iPSC-derived tissues from an unrelated (allogeneic) donor that express foreign human leucocyte antigens (HLA) may undergo immunological rejection requires the formulation of strategies to attenuate the host immune response to transplanted tissue. In clinical practice, individualized iPSC tissue derived from the intended recipient offers the possibility of personalized stem cell therapy in which graft rejection would not occur, but the logistics of achieving this on a large scale are problematic owing to relatively inefficient reprogramming techniques and high costs. The creation of stem cell banks comprising HLA-typed hESCs and iPSCs is a strategy that is proposed to overcome the immunological barrier by providing HLA-matched (histocompatible) tissue for the target population. Estimates have shown that a stem cell bank containing around 10 highly selected cell lines with conserved homozygous HLA haplotypes would provide matched tissue for the majority of the UK population. These simulations have practical, financial, political and ethical implications for the establishment and design of stem cell banks incorporating cell lines with HLA types that are compatible with different ethnic populations throughout the world.     

Therapeutic targets in cardiovascular disorders

The feasibility of gene therapy for cardiovascular diseases related to atherosclerosis is a topic that needs to be addressed. Most recent papers have dealt with technical aspects and feasibility and most of the genes transferred were reporter genes like those for β-galactosidase or luciferase. This may mean that the ideal vector, one that is not pathogenic or immunotolerant but is still efficient, is still not available. The results of these studies are ambiguous and it has been doubted whether the genes targeted really affect the disease. Further efforts are therefore needed to elucidate the underlying pathophysiology.     

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.     

Expression profile of the stem cell markers in human Hertwig’s epithelial root sheath/Epithelial rests of Malassez cells

Hertwig’s epithelial root sheath/Epithelial rests of Malassez (HERS/ERM) cells are unique epithelial cells in the periodontal ligament. They remain in periodontal tissues through-out the adult life, and it is expected that their functional role is to maintain the homeostasis of the periodontium through reciprocal interactions with other periodontal cells. In this study, we investigated whether HERS/ERM cells have primitive stem cell characteristics: those of embryonic stem cells as well as of epithelial stem cells. Primary HERS/ERM cells had typical epithelial cell morphology and characteristics and they maintained for more than five passages. They expressed epithelial stem cell-related genes: ABCG2, ANp63, p75, EpCAM, and Bmi-1. Moreover, the expression of embryonic stem cell markers such as Oct-4, Nanog, and SSEA-4 were detected. Next, we investigated whether the expression of these stem cell markers was maintained during the sub-culture process. HERS/ERM cells showed different expression levels of these stemness genes at each passage, but their expression was maintained throughout the passages. Taken together, our data suggest that a primary culture of HERS/ERM cells contains a population of primitive stem cells that express epithelial stem cell markers and embryonic stem cell markers. Furthermore, these cell populations were maintained during the sub-culturing process in our culture conditions. Therefore, our findings suggest that there is a strong possibility of accomplishing cementum tissue engineering with HERS/ERM cells.     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

体细胞重编程为多能干细胞的研究进展

体细胞通过重编程转变成其他类型的细胞,在再生医学方面具有重要的应用前景。细胞重编程的方法主要有体细胞核移植、细胞融合、细胞提取物诱导、限定因子诱导等,这些方法可以不同程度地改变细胞命运。最近,限定因子诱导的多能干细胞（induced pluripotent stem cell。iPS）为重编程提供了一种崭新的方法,不仅可以避免伦理争议,还提供了一种更为便利的技术,为再生医学开辟了新的天地;同时,iPS技术为研究基因表达调控、蛋白质互作、机体生长发育等提供了一个非常重要的研究手段。本文主要论述了体细胞重编程的方法及iPS细胞的进展、面临的问题和应用前景。     

新物种诱导多能干细胞的研究进展

胚胎干细胞（embryonic stem cells,ESC）在人类遗传病学研究、疾病模型建立、器官再生以及动物物种改良和定向变异等方面的地位是其他类型的细胞不可取代的。但是,由于实验技术和体外培养条件的限制,除了小鼠、恒河猴和人之外,大鼠、猪、牛、羊等其他哺乳动物的ES细胞系被证明很难获得。先后有多个研究小组报道了他们利用新兴的诱导多能干细胞（induced pluripotent stem cells,iPS细胞）技术成功建立大鼠和猪的iPS细胞系的研究成果。迄今为止,这两个物种是在未成功建立ES细胞系之前利用iPS技术建立多能干细胞系的成功范例。这些研究对于那些还未建立ES细胞的物种建立多能干细胞系提供了一种新的方案,也将给这些物种的胚胎干细胞的建立、基因修饰动物的产生以及人类医疗事业的促进和发展带来新的希望。     

Heterocyst formation in Anabaena

Heterocystous cyanobacteria grow as multicellular organisms with a distinct one-dimensional developmental pattern of single nitrogen-fixing heterocysts separated by approximately ten vegetative cells. Several genes have been identified that are required for heterocyst development and pattern formation. A key regulator, HetR, has been recently shown to be aserine-type protease.