Derivation characteristics and perspectives for mammalian pluripotential stem cells

Pluripotential stem cells have been derived in mice and primates from preimplantation embryos, postimplantation embryos and bone marrow stroma. Embryonic stem cells established from the inner cell mass of the mouse and human blastocyst can be maintained in an undifferentiated state for a long time by continuous passage on embryonic fibroblasts or in the presence of specific inhibitors of differentiation. Pluripotential stem cells can be induced to differentiate into all the tissues of the body and are able to colonise tissues of interest after transplantation. In mouse models of disease, there are numerous examples of improved tissue function and correction of pathological phenotype. Embryonic stem cells can be derived by nuclear transfer to establish genome-specific cell lines and, in mice, it has been shown that embryonic stem cells are more successfully reprogrammed for development by nuclear transfer than somatic cells. Pluripotential stem cells are a very valuable research resource for the analysis of differentiation pathways, functional genomics, tissue engineering and drug screening. Clinical applications may include both cell therapy and gene therapy for a wide range of tissue injury and degeneration. There is considerable interest in the development of pluripotential stem cell lines in many mammalian species for similar research interests and applications.     

Methods for the measurement of cell and tissue compatibility including tissue regeneration processes

Biocompatibility is one of the main requirements for the safe use of medical devices. Determination of cytotoxicity is part of the initial evaluation stipulated by ISO standards for the biological evaluation of medical devices. The use of cell cultures to test the biocompatibility of drugs, biomaterials or treatment techniques used in various disciplines is gaining in importance. A wide variety of self-initiated and commercially available cell lines has been evaluated and used: cultured fibroblasts from human skin, buccal mucosa, periodontal membrane, embryonic lung, epithelial and HeLa cells; cultures of human keratinocytes and HaCaT cells; different murine cell lines (C3H-L, Balb/c 3T3, L929 and others) as well as murine cells cultured from liver and spleen; T-lymphocytes from lymph nodes and macrophages obtained by lavage.All of the above cells are suitable for use in biocompatibility tests. Nevertheless, the general opinion is that toxicity tests in vitro will be more convincing when performed with cells that are homologous with the human tissue concerned. In accordance, appropriate cell lines for use in cytotoxicity and tolerance tests concerning the skin would be human dermal fibroblasts and human epidermal keratinocytes, as they take an active part in the immune response, inflammatory processes, and wound healing.The evaluation of the in vitro cytotoxicity of a biomaterial is often a qualitative analysis based on the morphological examination of cell damage and growth after direct or indirect contact with the material. Different commercial assays based on the determination of nucleic acids, metabolic activity, protein content or membrane integrity are available to measure cell proliferation and cell viability. A small selection – Pico Green^® DNA Cell Proliferation Assay, ATPLite™ Luminescence ATP Detection Assay, BC Assay: protein quantitation kit, AlamarBlue™ Proliferation Assay and Live/Dead Staining with SYTO-13 and EthD-2 – are discussed concerning sensitivity, reliability and applicability.     

Hepatocytes as feeder-layers for in vitro cultivation of Plasmodium falciparum blood-stages

To improve the in vitro growth of Plasmodium falciparum we attempted to cultivate its erythrocytic stages on monolayers of functionally active hepatocytes. Hepatocytes from Swiss Albino mice were isolated by perfusing the liver with a collagenase solution and were co-cultured with a liver epithelial cell type in RPMI 1640 medium supplemented with 10% human umbilical cord serum. The results show that the presence of hepatocytes improves both the multiplication rates of three strains of P. falciparum already in cultivation and the proliferation of freshly isolated strains. Of nine primary isolates tested, only three could be adapted in the standard conditions, whereas all grew readily in the presence of hepatocytes. After two to three weeks of culture with feeder cells, all the strains could be maintained continuously in standard conditions. Similar results were obtained using hepatocytes from another rodent species. Growth was also improved using the supernatant from hepatocyte cultures. No improvement resulted from the use of two human hepatoma cell lines, one rat hepatoma, human embryonic lung fibroblasts, human liver fibroblasts and rat liver epithelial cells as feeder layers. From these results it appears that better culture media can be designed and that the effect of hepatocytes is probably related to the specific functions exhibited by these cells. Hepatocytes may act either by removing toxic substances, particularly lactic acid in the Krebs and Cori cycles, and/or supplying nutrients essential to the parasite.     

两株人胚胎干细胞的无饲养层扩增

目的:建立两株中国人胚胎干细胞(hESCs)的无饲养层的培养体系,观察条件培养基所需的饲养层密度,并探索最佳的传代方式。方法:两株人胚胎干细胞hES-846XX和hES-1846XY分别使用不同密度(3×105/mL、6×105/mL和1.2×106/mL)获得的条件培养基(CM)在无饲养层培养体系培养12代以上,用机械分离法和Ⅳ型胶原酶法进行传代,观察比较结果并对所得hESCs鉴定。结果:6×105/mL～1.2×106/mL之间的密度都是合适密度;Ⅳ型胶原酶在长期传代中优于机械法传代;所得细胞维持干细胞未分化状态和全能性。结论:无饲养层的培养体系可以用于中国胚胎干细胞培养,两株中国人胚胎干细胞(hESCs)的倍增周期、所需饲养层密度及分化率等方面与国外hESCs有明显差别。     

The derivation and potential use of human embryonic stem cells

Human embryonic stem cells lines can be derived from human blastocysts at high efficiency (>50%) by immunosurgical isolation of the inner cell mass and culture on embryonic fibroblast cell lines. These cells will spontaneously differentiate into all the primary embryonic lineages in vitro and in vivo, but they are unable to form an integrated embryo or body plan by themselves or when combined with trophectoderm cells. They may be directed into a number of specific cell types and this enrichment process requires specific growth factors, cell-surface molecules, matrix molecules and secreted products of other cell types. Embryonic stem (ES) cells are immortal and represent a major potential for cell therapies for regenerative medicine. Their use in transplantation may depend on the formation of a large bank of suitable human leucocyte antigen (HLA) types or the genetic erasure of their HLA expression. Successful transplantation may also require induction of tolerance in recipients and ongoing immune suppression. Although it is possible to customize ES cells by therapeutic cloning or cytoplasmic transfer, it would appear unlikely that these strategies will be used extensively for producing ES cells compatible for transplantation. Embryonic stem cell research may deliver a new pathway for regenerative medicine.     

低剂量脉冲超声波对培养细胞增殖作用

目的 研究不同条件低剂量脉冲超声波刺激体外培养人皮肤成纤维细胞的增殖情况.方法 采用5-澳脱氧尿苷(BrdU)掺入法测定成纤维细胞增殖情况.结果 在BrdU掺入法测定中,细胞培养液不含小牛血清(FCS)时,超声波刺激时间为8和14min剂量组的BrdU阳性着色细胞率与对照组比较有显著性增高(P＜0.05,P＜0.01);而细胞培养液含10?S时,超声波刺激时间为5,8,11和14min剂量组的BrdU阳性着色细胞率与对照组比较.差异有统计学意义(P＜0.01).且培养液的FCS含量分别为0%,5%和10%时,BrdU阳性着色细胞率的高峰值均在低剂量脉冲超声波刺激时间为8～11min时.结论 在一定刺激时间内,低剂量脉冲超声波能促进人皮肤成纤维细胞增殖,其增殖情况与超声波的刺激时间以及细胞培养液中是否含有小牛血清有关.     

Vitamin E (d-alpha-tocopheryl succinate) decreases mitotic accumulation in gamma-irradiated human tumor, but not in normal, cells

Previous studies have shown that treatment of tumor cells in vitro with d-alpha-tocopheryl succinate (alpha-TS), a most effective form of vitamin E, alone or in combination with X-irradiation, reduced the growth of these cells more than that produced by individual agents. However, it is unknown whether alpha-TS, alone or in combination with gamma-irradiation, would produce similar effects on normal cells. To study this, we have compared the effects of alpha-TS on three human tumor cell lines, HeLa (cervical carcinoma), OVGI (ovarian carcinoma), and A549 (lung carcinoma), with the effects on three human normal fibroblast lines, GM2149, AG1522, and HF19. Results showed that alpha-TS treatment of HeLa cells for 20 hours caused inhibition of growth in a dose-dependent manner, but normal human fibroblasts treated similarly with alpha-TS did not show such an effect. alpha-TS treatment for 20 hours also decreased mitotic accumulation in all three tumor cell lines but did not produce such an effect in any of the normal fibroblasts. As expected, gamma-irradiation with 1 Gy decreased mitotic accumulation in human tumor cells and normal fibroblasts; however, alpha-TS treatment for 24 hours before, during, and after irradiation for the entire experimental period further decreased mitotic accumulation in human tumor cells but not in normal cells. These data suggest that effects of alpha-TS, alone or in combination with gamma-irradiation, are selective for tumor cells. Therefore, existing fear that antioxidants such as vitamin E may protect cancer cells from free radical damage during radiation therapy is not justified.     

Lack of an association between cellular phospholipid triene: tetraene ratio and proliferation of human skin fibroblasts in culture

Two experiments were performed in an attempt to establish an association between cellular phospholipid triene:tetraene ratio and proliferation of human neonatal skin fibroblasts in culture. In Experiment 1, a low lipid culture medium was developed that caused an accumulation of (n-9) eicosatrienoic acid in the phospholipids of human fibroblasts. This culture medium, when supplemented with a mixture of mitogens, supported growth of human fibroblasts at a level equivalent to that found under conditions of maximal growth using serum supplementation (8% fetal bovine serum). The triene:tetraene ratio of fibroblast phospholipids under the two conditions was 1.88 vs. 0.03, suggesting that the growth of these cells was not adversely affected by a high (greater than 0.4) triene: tetraene ratio. In Experiment 2, cells were cultured in a low lipid, mitogen-supplemented medium with 16:1(n-7), 18:1(n-9), 18:2(n-6) or 20:4(n-6) added as the albumin complex. All the fatty acids permitted an equivalent maximal growth stimulation in the assay system, although having different effects on the phospholipid triene:tetraene ratio. The results suggest that there is a lack of an association between cellular phospholipid triene:tetraene ratio (range, 0.03 to 3.4) and proliferation of human fibroblasts in this culture system.     

Forschen mit humanen embryonalen Stammzellen in Deutschland

From July 2002 to May 2008, 36 applications for the import and use of human embryonic stem cells (hES) were reviewed by the German Central Ethics Committee for Stem Cell Research (ZES). A flood of applications anticipated by opponents to human embryonic stem cell research has not occurred since the enactment of the German Stem Cell Act in 2002. On the contrary, German hES cell research is below international average in terms of project numbers. The current restrictions for using hES cells in Germany might be causative for the opinion that this type of research is not considered to be very promising. This could hold true especially for research aiming at clinical applications. Consequently, potential research goals of premium importance, especially those of potential clinical relevance, could be seriously jeopardized.     

Shifting paradigms? Reflections on regenerative medicine,embryonic stem cells and pharmaceuticals

Will human embryonic stem (hES) cells lead to a revolutionary new regenerative medicine? We begin to answer this question by drawing on interviews with scientists and clinicians from leading labs and clinics in the UK and the USA, exploring their views on the bench‐bedside interface in the fields of hES cells, neuroscience and diabetes. We employ Bourdieu's concepts of field, habitus and capital in order to understand stem cell science and cell transplantation. We also build on research on the sociology of expectations, and explore expectations of pharmaceutical approaches in hES research through our concept of ‘expectational capital’. In the process we discuss emerging expectations within stem cell research, most especially the ‘disease in a dish’ approach, where hES cells will be used as tools for unravelling the mechanisms of disease to enable the development of new drugs. We argue that experts’ persuasive promises advance their interests in the uncertain stem cell field, and explore how this performative strategy might stabilise the emerging ‘disease in a dish’ model of translational research.     

Therapeutic cloning in debate]

Human embryos can be conceived by cell nuclear transfer in order to isolate human embryonic stem cells (hES cells) for research into autologous cell therapy (therapeutic cloning). However, this technique broaches the major ethical problem concerning the instrumental use of human preimplantation embryos. From the viewpoint of subsidiarity, it is argued that various potential alternatives for therapeutic cloning should first be investigated further. The question as to whether therapeutic cloning should be allowed only becomes apparent when research with surplus embryos obtained in the course of in-vitro fertilization suggests that usable transplants can be obtained in vitro from hES cells, and when the potential alternatives for therapeutic cloning are either less promising or need more time for development than is currently expected.     

伏马菌素细胞毒性的研究进展

伏马菌素毒性作用机制尚未研究清楚,目前采用了广泛的细胞试验进行研究。本文介绍了几类用于此项研究的细胞试验,包括纤维原细胞、肠细胞、肝细胞、肾细胞、免疫细胞、星形胶质细胞、神经细胞。探讨了伏马菌素主要的细胞毒性作用及机制。     

Collagen synthesis in human skin fibroblasts is stimulated by a stable form of ascorbate, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid.

We evaluated the effect of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) on collagen synthesis in cultured human skin fibroblasts and on proliferation of fibroblasts. At concentrations of 0.1-0.5 mmol/L, AA-2G effectively stimulated collagen synthesis with an effectiveness comparable to that of L-ascorbic acid. On the other hand, 6-O-alpha-D-glucopyranosyl-L-ascorbic acid showed a weak effect. The stimulation of collagen synthesis by AA-2G was attenuated by the addition of a collagen synthesis inhibitor, L-azetidine 2-carboxylic acid, in a dose-dependent manner. In addition, AA-2G-induced stimulation of collagen synthesis could be completely inhibited by the addition of castanospermine, an inhibitor of neutral alpha-glucosidase. Relatively high alpha-glucosidase activity, which would contribute to release of ascorbic acid from AA-2G, could be detected in the lysate of cultured fibroblasts. The stimulatory activity of AA-2G on collagen synthesis was observed after 5 d in culture, whereas L-ascorbic acid tended to lose its stimulatory activity. Continuous supplementation of AA-2G (0.25 mmol/L) to culture medium for 24 d enhanced the cell growth four times that of the control. These results indicate that AA-2G is gradually cleaved by the cellular alpha-glucosidase to release L-ascorbic acid, which adequately stimulates collagen synthesis and proliferation of human skin fibroblasts.     

Human embryonic stem cells: challenges and opportunities

Human and non-human primate embryonic stem (ES) cells are invaluable resources for developmental studies, pharmaceutical research and a better understanding of human disease and replacement therapies. In 1998, subsequent to the establishment of the first monkey ES cell line in 1995, the first human ES cell line was developed. Later, three of the National Institute of Health (NIH) lines (BG01, BG02 and BG03) were derived from embryos that would have been discarded because of their poor quality. A major challenge to research in this area is maintaining the unique characteristics and a normal karyotype in the NIH-registered human ES cell lines. A normal karyotype can be maintained under certain culture conditions. In addition, a major goal in stem cell research is to direct ES cells towards a limited cell fate, with research progressing towards the derivation of a variety of cell types. We and others have built on findings in vertebrate (frog, chicken and mouse) neural development and from mouse ES cell research to derive neural stem cells from human ES cells. We have directed these derived human neural stem cells to differentiate into motoneurons using a combination of developmental cues (growth factors) that are spatially and temporally defined. These and other human ES cell derivatives will be used to screen new compounds and develop innovative cell therapies for degenerative diseases.     

脂多糖对人正常皮肤成纤维细胞增殖周期及Ⅰ、Ⅲ型前胶原mRNA表达的影响

目的观察大肠杆菌脂多糖（lipopolysaccharide,LPS）刺激后,皮肤成纤维细胞增殖周期和Ⅰ、Ⅲ型前胶原mRNA表达变化规律。方法体外培养正常人皮肤成纤维细胞,加入不同浓度（0．005～1．0μg／m1）的大肠杆菌LPS（Ecoli055：B5）,于LPS加入后第7天细胞处于对数生长期时,采用流式细胞术观察细胞增殖周期;用逆转录-聚合酶链反应（RT-PCR）法测定成纤维细胞Ⅰ、Ⅲ型前胶原mRNA及胶原酶mRNA的表达。结果LPS刺激浓度在0．005～0．1μg／ml时,S期细胞比例随着刺激浓度增加而增高,且呈浓度依赖性;当LPS浓度为0．5μg／ml时,上述作用开始降低,但仍高于空白对照;当浓度达到1．0μg／ml时,s期细胞比例均低于空白对照;LPS刺激浓度在0．005～0．1μg／ml时,促进正常皮肤成纤维细胞Ⅰ、Ⅲ型前胶原mRNA表达,抑制胶原酶mRNA表达,且呈一定的剂量依赖性;当LPS刺激浓度为0．5μg／ml,上述作用下降;而当LPS刺激浓度到达1．0μg/ml时,抑制正常皮肤成纤维细胞Ⅰ、Ⅲ型前胶原mRNA表达,促进胶原酶mRNA表达。结论在一定浓度范围内LPS促进皮肤成纤维细胞增殖和胶原合成,而过高浓度LPS则呈现抑制效应。     

转染人内皮抑素对卵巢癌细胞A2780体内外生长的影响

目的:探讨人内皮抑素(hum an endostatin,hES)对人卵巢癌细胞株A2780体内、外生长的影响及其可能机制。方法:高保真PCR从含有人内皮抑素基因的人肝脏组织中扩增出人内皮抑素编码区克隆入pcDNA3.1载体中,构建pcDNA3.1-ES重组质粒;将此重组质粒转染人卵巢癌细胞A2780中,观察A2780细胞生长。建立人卵巢癌裸鼠模型,随机将其分为两组:hES转染细胞组(A组,10只),对照组(B组,10只)。1月后,测量瘤体积、计算抑瘤率;标本行HE染色分析组织学形态;同时用W estern b lot检测hES、凋亡抑制蛋白bc l-2的表达;流式细胞仪检测转染对裸鼠移植瘤细胞凋亡率的影响。结果:hES转染A2780细胞后,细胞生长速率减慢。转染hES的裸鼠肿瘤生长较对照组明显减慢(P<0.001),抑瘤率为74%;HE染色及W estern b lot提示:转染后(A组)的组织学形态与转染前(B组)有显著差异;A组hES蛋白水平明显高于B组,而bc l-2蛋白水平明显低于B组;流式结果显示转染使肿瘤细胞的凋亡显著增加(P<0.05)。结论:转染hES体内、外均可抑制卵巢癌A2780细胞的生长,其机制与其促进了凋亡有关。     

Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses

Comparative studies were carried out on three mosquito cell lines (C6/36 clone of Aedes albopictus, AP-61 from A. pseudoscutellaris, and TRA-284 from Toxorhynchites amboinensis) to determine their sensitivity to dengue virus isolation, growth, and handling characteristics for immunofluorescent testing. Virus isolation rates from human sera were the highest in the TRA-284-SF (a line adapted to serum-free medium), followed by the TRA-284 parental line and AP-61. Virus isolation was the lowest in the C6/36 line. All 3 cell lines were comparable in terms of ease of handling, but C6/36 cells were preferable for detecting infected cells by the direct fluorescent antibody test (DFAT) because of frequent cell clumping in the AP-61 and TRA-284 lines. Early detection of viral antigen of all 4 serotypes in the infected cells by DFAT was dependent upon the virus titre in the serum. The AP-61 and TRA-284-SF cells were the best for early detection and identification of viral antigen. Similarly, both AP-61 and TRA-284 cells were more resistant than C6/36 cells to toxic effects of human sera. Based on the economy of using the serum-free medium, their higher sensitivity for dengue virus isolation, and their ease of handling, it is recommended that the TRA-284-SF cell line be used for routine dengue virus isolation in laboratories with cell culture capability.     

IDENTIFICATION OF MATURATION-RELATED WHEAT-GERM LECTIN-BINDING PROTEINS IN THE CULTURE OF HUMAN CORPUS EPIDIDYMAL EPITHELIAL CELLS

This study is designed to investigate the synthesis of maturation-related wheat germ agglutinin (WGA) binding glycoproteins in the human corpus epididymal epithelial cells by in vitro culture. Epithelial cells were isolated from the corpus of human epididymides and cultured with RPMI 1640 medium supplemented with 10% fetal calf serum in type IV collagen-coated dishes at 37°C. The epithelial nature, presence of fibroblasts, WGA-binding sites, and existence of GP-83 were determined by an indirect immunocytochemical and histochemical staining technique. Proteins in the cultured cells were analyzed by SDS-PAGE and autoradiography. After culturing for 10 days, the cells were shown to be positive with epithelial cell-specific keratins but devoid of fibroblasts. WGA-binding granules and positive binding sites of GP-83 were also detected in the cytoplasm. Immunoblots of cell extracts probed with the anti-GP-83 antibody from seminal fluid revealed the sperm maturation-related glycoprotein GP-83. The results indicate that WGA-binding proteins may be synthesized by the corpus epididymal epithelial cells of human and GP-83 may play an important role in sperm maturation. This culture model may be suitable for the investigation on the biosynthesis and physiology of human epididymal principal cells in vitro.     

肿瘤相关蛋白亚细胞结构定位的研究

目的应用激光共聚焦显微镜技术对肺癌相关蛋白N35进行亚细胞定位，观察它们在肿瘤细胞中的分布和肿瘤细胞周期不同时期的变化，并与其它组织和肿瘤进行比较，了解其特异性。方法利用抗人肺癌单克隆抗体N-35作为免疫探针作免疫荧光标记，应用激光共聚焦技术观察肿瘤相关蛋白亚细胞结构的定位，测定其相关抗原在肺癌细胞系GLC-82、人淋巴细胞系、人胚肺二倍体细胞系（KMB-17）、人皮肤纤维细胞系的存在及分布情况以及在肿瘤细胞周期不周时期的变化。结果肿瘤相关蛋白N35不存在于正常人淋巴细胞、正常肺细胞及人皮肤纤维细胞中，而存在于肺癌细胞蛋白中，在亚细胞结构中主要分布于胞核，在肿瘤细胞有丝分裂进入G2至M期时明确地定位于中心粒（centriole）结构上。结论肿瘤相关蛋白N35可能是一种只存在于肿瘤细胞并与其增殖活动密切相关的重要的肿瘤细胞生长调节蛋白，其功能可能与肿瘤细胞无限制增殖活动有关，是肿瘤标志物之一。可用于临床肿瘤的早期诊断、疗效评估、监测复发、预测转移和推测预后。     

Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media

Vero cells are nowadays widely used in the production of human vaccines. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective.The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated.In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspension-adapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 × 10⁶ cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers.Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ± 0.5 × 10⁵ cells/mL resulted in a virus titer higher than 10⁷ FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ± 0.5 × 10⁷ FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca⁺⁺ and Mg⁺⁺. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.