Trout sertoli and leydig cells: Isolation,separation, and culture

Trout testes at various stages of maturation were dissociated by perfusion at 12°C with collagenase plus pronase and then with collagenase alone, followed by slight shaking overnight in 1% bovine albumin. This step provided a suspension of isolated somatic and germ cells, clusters of interstitial cells, and either intact spermatogenetic cysts (meiotic testes) or clusters of Sertoli cells (other testes). Most of the spermatozoa were removed from the testis cell suspension by centrifugation in Percoll (density 1.065 g/ml). Sertoli and Leydig cells were prepared by a two-step separation method: (1) the testis cell suspension was separated by sedimentation at unit gravity into “isolated cell” and “cell cluster” populations; (2) these populations were fractionated by isopyknic centrifugation in Percoll gradients. In terms of somatic cell composition, a nearly pure Sertoli cell (clusters) population was obtained between 1.017 and 1.033 g/ml and a Leydig cell (clusters) enriched population of between 1.033 and 1.048 g/ml (testes resuming spermatogenesis) or 1.048 and 1.062 g/ml (other testes). These various cell populations were cultured in modified Leibovitz L15 medium for 10–15 days. When seeded, the Sertoli cells had a normal ultrastructure that remained unchanged for at least 10 days, and the steroidogenic activity of Leydig cells could be stimulated by salmon gonadotropin. Leydig cells remained 3β-HSD positive and produced progesterone and 17α, 20β-OH progesterone for at least 11 days. This study points out that viable and differentiated trout somatic testicular cells can be prepared and cultured for several days.     

BMP signaling and stem cell regulation

Stem cells play an essential role in cellular specialization and pattern formation during embryogenesis and in tissue regeneration in adults. This is mainly due to a stem cell's ability to replenish itself (self-renewal) and, at the same time, produce differentiated progeny. Realization of these special stem cell features has changed the prospective of the field. However, regulation of stem cell self-renewal and maintenance of its potentiality require a complicated regulatory network of both extracellular cues and intrinsic programs. Understanding how signaling regulates stem cell behavior will shed light on the molecular mechanisms underlying stem cell self-renewal. In this review, we focus on comparing the progress of recent research regarding the roles of the BMP signaling pathway in different stem cell systems, including embryonic stem cells, germline stem cells, hematopoietic stem cells, and intestinal stem cells. We hope this comparison, together with a brief look at other signaling pathways, will bring a more balanced view of BMP signaling in regulation of stem cell properties, and further point to a general principle that self-renewal of stem cells may require a combination of maintenance of proliferation potential, inhibition of apoptosis, and blocking of differentiation.     

干细胞理论及研究进展

干细胞是目前细胞工程研究最活跃的领域,通过对各种干细胞的界定,胚胎干细胞和成体干细胞的比较研究、以及干细胞的技术应用,揭示出干细胞尤其是胚胎干细胞在医学以及整个生命科学中的巨大潜势,乃至于引发医学领域的重大变革。     

微囊化K562细胞生长周期及代谢特性的研究

以K562细胞为模型,分别进行微囊化和游离培养,运用流式细胞术考察两种培养体系下细胞周期和生长代谢变化;建立数学模型,模拟了两种培养体系下细胞的生长活性和代谢特性。实验发现：微囊化培养过程中的K562细胞处于DNA合成期（S期）的百分含量显著高于游离培养,并且细胞保持较高的增殖活性。模型计算表明,所建模型动力学参数能够很好地描述微囊化和游离两种培养体系下细胞的代谢情况;对细胞活性的理论计算表明,微囊化的细胞具有较高的增殖和代谢活性,同时细胞能够较长时间保持此活性;模型参数表明,两种培养体系下,葡萄糖对细胞生长的影响无显著差别 (k^Free_L≈k^APA_L),乳酸对游离培养细胞的生长具有明显抑制作用,但对微囊化培养细胞抑制作用较小(kFreeL>≈kAPAL)。     

Stem cells and regenerative medicine

Stem cells have been defined as clonogenic cells that undergo both self-renewal and differentiation to more committed progenitors and functionally specialized mature cells. Of late years, stem cells have been identified in a variety of tissues of an adult body. Depending on the source, they have the potential to form one or more, or even all cell types of an organism. Stem cell research provided some outstanding contributions to our understanding of developmental biology and offered much hope for cell replacement therapies overcoming a variety of diseases. The establishment of human ES cell lines enabled us to generate all tissues we comprise. Recently, excitement has been evoked by the controversial evidence that adult stem cells have a much higher degree of developmental plasticity than previously imagined. More recently, the existence of multipotent somatic stem cells in bone marrow has been reported. Combined with these discoveries and achievements as well as the developing technologies, scientists are now trying to bring stem cell therapies to the clinic.     

钙离子对鼠角质细胞生长和分化的影响

用无血清培养基培养角质细胞,研究了Ca²⁺对鼠角质细胞生长和分化的影响。实验结果表明,培养基中钙离子最佳浓度为0.2mmol/L。在此浓度下,细胞克隆形成率达到10.8%,细胞的贴壁率达到28.7%,细胞的分化比例和老化比例分别为5.4%和26.3%;当Ca²⁺浓度达到0.6mmol/L以上时,则会引起角质细胞显著的分化和老化。     

Zinc and immunity

Nutritional deficiency of zinc is widespread throughout the developing countries and a conditioned deficiency of zinc is known to occur in many diseased states. Zinc is known to play an important role in the immune system and zinc deficient subjects may experience increased susceptibility to a variety of pathogens. We have studied the effects of a mild deficiency of zinc on T cells in an experimental model of human zinc deficiency. We showed that T cell functions were affected adversely even when the deficiency of zinc was mild in humans. Characteristically during zinc deficiency, the serum thymulin activity (a thymic hormone) was decreased which was restored following zinc supplementation. Our studies also showed that zinc deficiency caused an imbalance between TH1 and TH2 functions. The production of IFN-g, IL-2, TNF-a (products of TH1 cells) were decreased, whereas the production of IL-4, IL-6 and IL-10 (products of TH2) were not affected during zinc deficiency. T cell subpopulation studies revealed that the CD4+ CD45RA+ to CD4+ CD45RO+ ratio was decreased as a result of zinc deficiency, suggesting that zinc may be required for the regeneration of new CD4+ T cells. We further documented that zinc deficiency decreased NK cell lytic activity and caused a decrease in the percentage of CD8+ CD73+ T cells which are known to be predominantly precursors of cytotoxic T cells. In a suitable cell culture model our studies revealed that the gene expression of a DNA synthesizing enzyme TK was affected adversely which resulted in delayed cell cycle and decreased cell growth. The above immunological consequences of zinc deficiency may be responsible for decreased cell mediated immune functions in zinc deficient subjects.     

牛脂肪间充质干细胞的分离、培养与鉴定

为了给组织工程提供种子细胞,对牛间充质干细胞(Adipose-derived stem cells,ADSCs)进行体外分离培养。首先应用胶原酶消化法分离牛ADSCs,进行体外培养、连续传代,并观察细胞的形态变化,通过细胞计数绘制生长曲线,细胞压片进行染色体分析,采用细胞免疫荧光化学方法检测细胞表面标记,利用成骨分化和成脂分化检测其分化能力。结果显示牛ADSCs体外培养时细胞形态呈成纤维细胞样,增殖稳定;Vimentin、CD49d、CD13表达呈阳性,CD34表达呈阴性;成骨诱导条件下的细胞碱性磷酸酶活性高,茜素红染色呈阳性;成脂诱导条件下细胞周围脂滴明显,油红-O染色呈阳性。结果证明牛ADSCs体外生长稳定、增殖速度快、定向分化能力强,简易的体外分离培养及诱导方法为其在组织工程中的应用奠定了基础。     

Of mice, frogs and flies: generation of membrane asymmetries in early development

Embryonic development begins with cleavage of the fertilized egg. Cleavage comprises two major processes: cytokinesis and formation of a polarized epithelial cell layer. The focus of this review is comparison of the generation of membrane polarity during embryonic cleavage in three different developmental model systems. In mammalian embryos, as exemplified by analysis of the mouse, generation of distinct membrane domains is uncoupled from cleavage divisions and is initiated in a specific developmental phase, called compaction. In Xenopus laevis embryos, generation of polarized blastomeres occurs simultaneously with cytokinesis. The origin of specific membrane domains of X. laevis polar blastomeres, however, can be traced back to oogenesis. Finally, in Drosophila melanogaster, generation of polarized cells occurs at cellularization. The relevance of cell adhesion, cell junctions and cytocortical scaffolds will be discussed for each of the model systems. Despite enormous morphologic differences, the three models share many common features; in particular, many important molecular interactions are conserved.     

Efficient and Innocuous Live‐Cell Delivery: Making Membrane Barriers Disappear to Enable Cellular Biochemistry

The confluence of protein engineering techniques and delivery protocols are providing new opportunities in cell biology. In particular, techniques that render the membrane of cells transiently permeable make the introduction of nongenetically encodable macromolecular probes into cells possible. This, in turn, can enable the monitoring of intracellular processes in ways that can be both precise and quantitative, ushering an area that one may envision as cellular biochemistry. Herein, the author reviews pioneering examples of such new cell‐based assays, provides evidence that challenges the paradigm that cell penetration is a necessarily damaging and stressful event for cells, and highlights some of the challenges that should be addressed to fully unlock the potential of this nascent field.     

The role of glutamine, serum and energy factors in growth of enterocyte-like cell lines

Background: Glutamine is routinely added to most cell cultures. Glutamine has been found to be the preferential nutrient to the rapidly replicating intestinal mucosa, but whether this is a metabolic effect or due to other properties of this amino acid is not determined. To study the importance of glutamine on the growth of two enterocyte-like cell lines, the effects of depriving the media or supplementing it with glutamine were assessed in media with different serum and energy supplements. Methods: CaCo-2 and HT-29 cells were grown in serum-free medium, with fetal bovine or synthetic serum, and with or without glucose or galactose. The glutamine content was varied between 0 and 4 mM. All growth assays were performed in triplicate by counting in a hemocytometer. Results: Both cell lines were dependent of serum factors for growth, but displayed distinct requirements on glutamine supplementation. Glutamine was an obligate supplement with dose-dependent correlation to growth (r=0.87, p<0.01) for CaCo-2 cells cultured in synthetic, but not in fetal bovine serum. In HT-29 cells, the correlation between glutamine and growth was significant (r=0,68, p<0,05) only in fetal bovine serum in the absence of galactose. Conclusion: This study shows that glutamine has different growth stimulating effects on two enterocyte-like cell lines studied. This could reflect different modes of action of glutamine on proliferation and differentiation in an enterocyte cell population.     

Ultrastructural evidence for two-cell and three-cell neural pathways in the tentacle epidermis of the sea anemone Aiptasia pallida.

Sensory and ganglion cells in the tentacle epidermis of the sea anemone Aiptasia pallida were traced in serial transmission electron micrographs to their synaptic contacts on other cells. Sensory cell synapses were found on spirocytes, muscle cells, and ganglion cells. Ganglion cells, in turn, synapsed on sensory cells, spirocytes, muscle cells, and other neurons and formed en passant axo-axonal synapses. Axonal synapses on nematocytes and gland cells were not traced to their cells of origin, i.e., identified sensory or ganglion cells. Direct synaptic contacts of sensory cells with spirocytes and sensory cells with muscle cells suggest a local two-cell pathway for spirocyst discharge and muscle cell contraction, whereas interjection of a ganglion cell between the sensory and effector cells creates a local three-cell pathway. The network of ganglion cells and their processes allows for a through-conduction system that is interconnected by chemical synapses. Although the sea anemone nervous system is more complex than that of Hydra, it has similar two-cell and three-cell effector pathways that may function in local responses to tentacle contact with food.     

Cytotoxicity and binding profiles of activated Cry1Ac and Cry2Ab to three insect cell lines

While Cry1Ac has been known to bind with larval midgut proteins cadherin, APN (amino peptidase N), ALP (alkaline phosphatase) and ABCC2 (adenosine triphosphate‐binding cassette transporter subfamily C2), little is known about the receptors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baseline cytotoxicity of activated Cry1Ac and Cry2Ab against the midgut and fat body cell lines of Helicoverpa zea and the ovary cell line of Spodoptera frugiperda (SF9). As expected, the descending order of cytotoxicity of Cry1Ac against the three cell lines in terms of 50% lethal concetration (LC₅₀) was midgut (31.0 μg/mL) > fat body (59.0 μg/mL) and SF9 cell (99.6 μg/mL). By contrast, the fat body cell line (LC₅₀ = 7.55 μg/mL) was about twice more susceptible to Cry2Ab than the midgut cell line (16.0 μg/mL), the susceptibility of which was not significantly greater than that of SF9 cells (27.0 μg/mL). Further, ligand blot showed the binding differences between Cry1Ac and Cry2Ab in the three cell lines. These results indicated that the receptors of Cry2Ab were enriched in fat body cells and thus largely different from the receptors of Cry1Ac, which were enriched in midgut cells.     

Spindle orientation and epidermal morphogenesis

Asymmetric cell divisions (ACDs) result in two unequal daughter cells and are a hallmark of stem cells. ACDs can be achieved either by asymmetric partitioning of proteins and organelles or by asymmetric cell fate acquisition due to the microenvironment in which the daughters are placed. Increasing evidence suggests that in the mammalian epidermis, both of these processes occur. During embryonic epidermal development, changes occur in the orientation of the mitotic spindle in relation to the underlying basement membrane. These changes are guided by conserved molecular machinery that is operative in lower eukaryotes and dictates asymmetric partitioning of proteins during cell divisions. That said, the shift in spindle alignment also determines whether a division will be parallel or perpendicular to the basement membrane, and this in turn provides a differential microenvironment for the resulting daughter cells. Here, we review how oriented divisions of progenitors contribute to the development and stratification of the epidermis.     

Integrins and dystroglycan regulate astrocyte wound healing: the integrin beta1 subunit is necessary for process extension and orienting the microtubular network

Monolayers of astrocytes in culture respond to a scrape wound by orienting towards the wound and extending processes that will repair it. We show here that they also upregulate the expression of extracellular matrix (ECM) proteins, laminin, and chondroitin sulfated proteoglycan, that are deposited in astrocytic scars in vivo. We have previously shown that the major functional ECM receptors on astrocytes are dystroglycan (DG) plus integrins alpha1beta1, alpha5beta1, alpha6beta1, and alphavbeta3. Consistent with this, laminin fragments that activate alpha1beta1 integrin, alpha6beta1 integrin, and DG all contribute to attachment. During astrocyte attachment, or process extension, integrins and DG are found at the leading edge of the lammelipodium, though they change in distribution with the extent of attachment and the alpha and beta subunits of DG can be spatially uncoupled. Functionally, inhibitory antibodies to DG and integrin alpha1beta1 or the RGD peptide all inhibit process extension, showing that ligand engagement of integrins and DG contribute to process extension. Astrocytes differentiated from DG or beta1 null ES cells respond very differently to wounding. The former fail to extend process and cell polarization is disrupted partially. However, beta1 null astrocytes not only fail to extend processes perpendicular to the wound, but cell polarization is completely disrupted and cells migrate randomly into the wound. We conclude that integrins are essential for astrocyte polarity.     

Nuclear migration, nucleokinesis and lissencephaly

During the past 20 years, biologists have become used to finding that proteins first identified in simple, genetically manipulable eukaryotic organisms are conserved in higher eukaryotes. This article draws attention to the similarity between NUDF protein, which is required for nuclear migration in the filamentous fungus Aspergillus nidulans, and a mammalian homologue, LIS1, whose malfunction causes lissencephaly, a neuronal migration disease. The authors suggest that there might be an underlying similarity of mechanism between nuclear migration in the fungus and neuronal migration in the brain.     

被子植物生殖细胞和精细胞

随着被子植物精细胞分离技术的突破和细胞生物学以及分子生物学技术的发展,对被子植物精细胞的研究不断深入。在以前细胞生物学研究的基础上结合近年来的分子生物学研究结果对被子植物雄性生殖细胞的产生、精细胞的形成和发育以及有关精细胞的表面蛋白质、精细胞的特异启动子、精细胞cDNA文库的构建等分子生物学研究进展和今后的发展趋势进行了综述。     

To The Abercrombie Meeting and back again

The 7th Abercrombie Meeting took place in Oxford this past summer. It was organized by The Royal Microscopical Society with the support of The British Society for Cell Biology. Michael Abercrombie was a pioneer in the field of investigating cell behavior using time-lapse microscopy. The meeting was focused on “multi-dimensional cell migration in development and disease” and it brought together many of the world's leading researchers in the area, providing an opportunity to discuss the very latest advances and possible future developments in the field. The meeting sessions included Invasive Migration, Invasive Adhesions in Migrating Cells, Signaling in Migration, Immune Cell Migration, Migrations during Morphogenesis and Migration and Disease. As with all Abercrombie meetings, the conference delegates were treated to a staggering array of live cell imaging, in vivo imaging and images generated by the latest developments in microscopy.     

Terminology and classification of stomata and stomatal development—a critical survey

The literature on terminology of stomata and stomatal development is reviewed and the terminology rationalized. The classification of developmental types and of the developing cells should not be combined with the morphological classification of mature stomatal complexes. The cells involved in the development should be distinguished on the basis of their origin and position in the developing stomatal complex, and not on the basis of their future form and appearance. It is unsound to distinguish any kind of cell only on the basis of a presumed future division by which it is replaced by its two daughter cells. Development of stomata begins with the formation of a stomatal meristemoid by an unequal division of a protodermal cell. A meristemoid may divide unequally to produce a new meristemoid and a mesogene cell. Stomatal meristemoids eventually function as guard-cell mother-cells. The adjective perigene is restricted to those cells that have arisen by divisions of protodermal cells surrounding the future stoma. The undivided cells surrounding protodermal cells should be termed agene cells, and not neighbouring cells, a term which should be restricted to morphological terminology.