Oxidative stress-induced biomarkers for stem cell-based chemical screening

Stem cells have been considered for their potential in pharmaceutical research, as well as for stem cell-based therapy for many diseases. Despite the potential for their use, the challenge remains to examine the safety and efficacy of stem cells for their use in therapies. Recently, oxidative stress has been strongly implicated in the functional regulation of cell behavior of stem cells. Therefore, development of rapid and sensitive biomarkers, related to oxidative stress is of growing importance in stem cell-based therapies for treating various diseases. Since stem cells have been implicated as targets for carcinogenesis and might be the origin of “cancer stem cells”, understanding of how oxidative stress-induced signaling, known to be involved in the carcinogenic process could lead to potential screening of cancer chemopreventive and chemotherapeutic agents. An evaluation of antioxidant states reducing equivalents like GSH and superoxide dismutase (SOD), as well as reactive oxygen species (ROS) and nitric oxide (NO) generation, can be effective markers in stem cell-based therapies. In addition, oxidative adducts, such as 4-hydroxynonenal, can be reliable markers to detect cellular changes during self-renewal and differentiation of stem cells. This review highlights the biomarker development to monitor oxidative stress response for stem cell-based chemical screening.     

大鼠胚胎脑新皮质神经干细胞的体外分离培养方法研究

目的建立大鼠胚胎脑新皮质神经干细胞的体外分离培养方法。方法从孕14天SD大鼠胚胎脑分离新皮质,通过机械吹打改善细胞分散状态、优化无血清培养基条件,从而改进神经干细胞的培养方法。利用神经干细胞标志蛋白(nestin和SOX2)、增殖和克隆成球能力以及多向分化能力测试三方面对神经干细胞进行鉴定。结果以该法培养的神经干细胞nestin蛋白呈强阳性表达,SOX2阳性表达率达99%以上,BrdU掺入阳性,培养3天后细胞量为接种量的10.55倍,6日克隆成球率为(33.00±4.40)%。经相应特异性标志蛋白检测证实神经干细胞经诱导分化后可形成神经元(MAP2)、星形胶质细胞(GFAP)和少突胶质细胞(O4)。结论获得的神经干细胞纯度和细胞产出率高,具有强自我更新和多向分化能力。     

Mesenchymal stem cell; history, biology and clinical application

In the last years, stem cells have drawn the attention of various sectors of society for many reasons. From the basic point of view, stem cells represent an ideal model to study cell differentiation and self-renewal mechanisms. However, their potential in cell therapy and regenerative medicine has triggered the increasing amount of knowledge in this area. Mesenchymal stem cells belong to the select group of adult stem cells. They have differentiation potential towards mesenchymal tissues such as bone, cartilage, stroma and fat. Recently, both in vivo and in vitro reports have shown a greater plasticity of mesenchymal stem cells, showing not only a mesenchymal cell fate but also those leading to endothelial, nervous and muscular lineages. For these reasons, the study of mesenchymal stem cells has gained great interest and many articles have been published. In the present review, we have presented a global vision of this topic, including its history, biologic features, sources, isolation methods and an overview on their clinical application.     

Zinc and neurogenesis: making new neurons from development to adulthood

Stem cell proliferation, neuronal differentiation, cell survival, and migration in the central nervous system are all important steps in the normal process of neurogenesis. These mechanisms are highly active during gestational and early neonatal brain development. Additionally, in select regions of the brain, stem cells give rise to new neurons throughout the human lifespan. Recent work has revealed key roles for the essential trace element zinc in the control of both developmental and adult neurogenesis. Given the prevalence of zinc deficiency, these findings have implications for brain development, cognition, and the regulation of mood.     

Enrichment of blood from embryonic stem cells in vitro

Murine embryonic stem (ES) cells are pluripotent. When injected into blastocysts they can give rise to every cell type of a derived chimeric mouse including germ cells. Embryonic stem cells also possess remarkable in vitro differentiation potential. When removed from stromal support and leukaemia inhibitory factor (LIF), ES cells differentiate into structures known as embryoid bodies (EBs), in which all three germ layers develop and interact. As ES cells from humans become available there is increasing interest in the potential for EBs to provide an unlimited supply of stem cells for somatic transplantation therapies. Realisation of this potential will require greater understanding of the molecular determinants of cell fate within EBs. Also, culture techniques for selective expansion of cell lineages of interest will reduce the risks associated with transplantation of EB-derived cells. In this paper the kinetics of expression of mRNA and protein for early mesoderm markers within EBs is reported. In addition, a three-step culture system incorporating co-cultivation on the bone marrow derived stromal cell line, MC3T3-G2/PA6 (PA6), is explored as a way to select for haematopoietic progenitor cells (HPCs) and against undifferentiated ES cells. A system like this could enhance purification of haematopoietic stem cells (HSCs) from ES cells for bone marrow transplantation.     

Glutamine depletion disrupts mitochondrial integrity and impairs C2C12 myoblast proliferation,differentiation, and the heat-shock response

Glutamine and glucose are both oxidized in the mitochondria to supply the majority of usable energy for processes of cellular function. Low levels of plasma and skeletal muscle glutamine are associated with severe illness. We hypothesized that glutamine deficiency would disrupt mitochondrial integrity and impair cell function. C2C12 mouse myoblasts were cultured in control media supplemented with 5.6 mmol/L glucose and 2 mmol/L glutamine, glutamine depletion (Gln⁻) or glucose depletion (Glc⁻) media. We compared mitochondrial morphology and function, as well as cell proliferation, myogenic differentiation, and heat-shock response in these cells. Glc⁻ cells exhibited slightly elongated mitochondrial networks and increased mitochondrial mass, with normal membrane potential (ΔΨm). Mitochondria in Gln⁻ cells became hyperfused and swollen, which were accompanied by severe disruption of cristae and decreases in ΔΨm, mitochondrial mass, the inner mitochondrial membrane remodeling protein OPA1, electron transport chain complex IV protein expression, and markers of mitochondrial biogenesis and bioenergetics. In addition, Gln⁻ increased the autophagy marker LC3B-II on the mitochondrial membrane. Notably, basal mitochondrial respiration was increased in Glc⁻ cells as compared to control cells, whereas maximal respiration remained unchanged. In contrast, basal respiration, maximal respiration and reserve capacity were all decreased in Gln⁻ cells. Consistent with the aforementioned mitochondrial deficits, Gln⁻ cells had lower growth rates and myogenic differentiation, as well as a higher rate of cell death under heat stress conditions than Glc⁻ and control cells. We conclude that glutamine is essential for mitochondrial integrity and function; glutamine depletion impairs myoblast proliferation, differentiation, and the heat-shock response.     

Circulating stem cells and tissue repair

Stem cells are defined as cells that have clonogenic, self-renewing capacities and the capability to differentiate into multiple cell lineages. Whereas embryonic stem cells are derived from mammalian embryos in the blastocyst stage and can generate terminally differentiated cells of all 3 embryonic germ layers, adult human stem cells are capable of maintaining, generating, and replacing terminally differentiated cells within their own specific tissue as a consequence of physiologic cell turnover or tissue injury. The traditional idea of organ-restricted stem-cell differentiation is now being challenged by the suggestion that adult stem cells retain developmental plasticity. Preclinical and clinical studies described in this review provide evidence that within the blood circulate not only progenitor cells that differentiate into hematopoietic cells, but also stem/progenitor cells which can participate in the homeostasis, repair and replacement of solid organ tissues. In addition to the occurrence of cell fusion, there are 4 suggested mechanisms of adult stem cell differentiation into solid organ cells. Preclinical data support these models particularly that of transdifferentiation as the most likely model, allowing stem/progenitor cells to differentiate across lineage, tissue, and germ layer boundaries. There is increasing evidence that we can manipulate in vivo circulating adult stem cells to repair or regenerate solid organ tissue, which offers potential clinical benefit in the treatment of many hereditary and acquired diseases.     

人肺腺癌肿瘤细胞免疫表型CD133、CD34、CD44的实验研究

目的 拟验证CD133、CD34、CD44作为人肺腺癌肿瘤干细胞表面标记物的合理性.方法 采集新鲜肺腺癌组织标本,利用两种体外培养方法扩增出贴壁细胞和悬浮细胞球两种肿瘤细胞,采用免疫荧光检测比较CD133、CD34和CD44在两种培养细胞中表达的差异.结果 悬浮球肿瘤细胞培养较贴壁肿瘤细胞生长速度慢、维持时间长且成功率高(72.5% vs 47.5%,P＜0.05).CD133、CD34和CD44在悬浮细胞球中表达率和表达量明显高于贴壁肿瘤细胞(68.97%,82.76%,93.10% vs 5.26%,15.79%,5.26%,P＜0.01).结论 CD133、CD34和CD44可能作为分离人肺腺癌肿瘤干细胞的表面蛋白表标记组合.

Abstract：

Objective To validate the possibility of CD133 CD34 CD44 be served as biomarkers in cancer stem cell of human lung adenocarcinoma. Methods Two kinds of culturing methods were performed to generate adhesive tumor cells and floating aggregates, and the differences of expression of CD133 CD34 CD44 between 2 kinds of cultured cells were observed by immunofluorescence. Results Floating aggregates grew more slowly, kept activity for longer period than adhesive cells (72.5% vs 47.5%,P＜0.05). Floating aggregates expressed higher level of CD133, CD34 and CD44 than adhesive cells (68.97%,82.76%,93.10% vs 5.26%,15.79%,5.26%,P＜0.01). Conclusions The combination of CD133, CD34 and CD44 probably can be used as surface markers of cancer stem cells for human lung adenocarcinoma.     

干细胞与环境毒理学Ё

近年来,干细胞由于其在生物医药方面的巨大应用前景而成为科学研究的新热点。干细胞具有自我更新的能力,可以在体外进行培养并持续保持未分化状态,在一定条件下能够分化为机体的各种组织器官。因此干细胞正逐渐成为研究环境毒物对机体损伤的新工具,通过建立干细胞体外模型和体内组织干细胞研究相结合的方法可以进行环境毒物的早期危险度评测,寻找新的生物标志物,明确环境中的各种物质对机体的影响机制。本文综述报道各类干细胞及其在环境毒理学研究中的应用近况。     

Embryo implantation and embryonic stem cell development in primates

The endocrine dialogue that results in implantation and the successful establishment of pregnancy in primates relies on embryonic secretion of chorionic gonadotrophin (CG). This hormone is a signal of embryo viability and capacity to support the corpus luteum. The expression of CG is apparently restricted to primates. Active or passive immunization of marmoset monkeys against the beta subunit of CG prevented implantation and early pregnancy, without disrupting the ovarian cycle. Studies of individual embryos cultured in vitro showed that CG is secreted at low levels by the blastocyst from before attachment, with secretion increasing exponentially after attachment. Gonadotrophin releasing hormone (GnRH) was also secreted, from mid-blastocyst stages, before the detection of CG. The secretion of GnRH by the embryo continued through the attachment and outgrowth stages of embryonic differentiation in vitro. The hypothetical role of GnRH in regulating CG release during implantation was tested in recently completed experiments. Individual embryos cultured with GnRH, or with agonist or antagonist to GnRH, showed significant variations in their secretion of CG and in their survival in culture, suggesting a causal relationship between these hormones. Embryos cultured with natural GnRH showed enhanced growth and development. Embryonic stem cells, from the inner cell mass of marmoset and rhesus monkeys, were the first primate embryonic stem cells to be isolated and characterized, enabling the subsequent isolation of human embryonic stem cells.     

Stem cells and lineage development in the mammalian blastocyst

The mammalian blastocyst is the source of the most pluripotent stem cells known: embryonic stem (ES) cells. However, ES cells are not totipotent; in mouse chimeras, they do not contribute to extra-embryonic cell types of the trophectoderm (TE) and primitive endoderm (PrE) lineages. Understanding the genetic pathways that control pluripotency v. extra-embryonic lineage restriction is key to understanding not only normal embryonic development, but also how to reprogramme adult cells to pluripotency. The trophectoderm and primitive endoderm lineages also provide the first signals that drive patterned differentiation of the pluripotent epiblast cells of the embryo. My laboratory has produced permanent mouse cell lines from both the TE and the PrE, termed trophoblast stem (TS) and eXtra-embryonic ENdoderm (XEN) cells. We have used these cells to explore the genetic and molecular hierarchy of lineage restriction and identify the key factors that distinguish the ES cell v. the TS or XEN cell fate. The major molecular pathways of lineage commitment defined in mouse embryos and stem cells are probably conserved across mammalian species, but more comparative studies of lineage development in embryos of non-rodent mammals will likely yield interesting differences in terms of timing and details.     

异氟醚通过激活InsP_3受体对ReNcell CX人神经干细胞损伤和存活的双向调节作用

目的：研究InsP3受体在吸入性麻醉剂异氟醚对ReNcell CX人神经干细胞损伤和存活的影响中的作用。方法：将培养的ReNcell CX人神经干细胞应用2.6%异氟醚分别处理1h、4h、8h和12h后,测量LDH和MTT,研究异氟醚对ReNcell CX人神经干细胞存活的时间效应;在异氟醚处理前加入InsP3受体特异性阻滞剂100nMXc,再测量LDH和MTT,研究阻断InsP3受体能否消除异氟醚对ReNcell CX人神经干细胞存活的影响。结果：2.6%异氟醚处理1h,LDH低于对照组,MTT高于对照组;2.6%异氟醚处理12h,LDH高于对照组,MTT低于对照组;加入InsP3受体阻滞剂Xc后,再用2.6%异氟醚处理1h及12h,LDH和MTT与对照组差异无统计学意义。结论：异氟醚对ReNcellCX人神经干细胞损伤和存活具有双向作用,这种作用通过内质网膜上InsP3受体介导。     

5-氮杂胞苷对大鼠骨髓间充质干细胞增殖与分化的影响

目的:探讨5-氮杂胞苷(5-azacytidine,5-aza)对体外骨髓间充质干细胞(Mesenchymal stem cells,MSCs)细胞增殖与分化潜能的影响。方法:从Wistar大鼠骨髓中获得MSCs,培养传代,透射电镜下观察诱导前后细胞形态变化;MTT法检测5-aza对MSCs生长的影响;免疫细胞化学方法检测Actin(α-Sarcomeric)、Desmin、Myoglobin、NSE、GFAP的表达。结果:5-aza对MSCs有抑制作用,在透射电镜下观察可见有心肌样细胞和神经元样细胞的形态变化。免疫细胞化学检测结果表明,MSCs经5-aza诱导14dactin、desmin、myoglobin、NSE和GFAP的表达均较对照组显著增高,P0.01。结论:5-aza对MSCs细胞生长有抑制作用,MSCs被5-aza诱导分化成心肌样细胞和神经元样细胞。     

In vitro effects of melatonin on colonization of neonate mouse spermatogonial stem cells

We have recently reported that antioxidant supplements enhance the efficacy of cryopreserved spermatogonial stem cells. Melatonin is considered a free radical scavenger which has direct and indirect antioxidant effects in in vitro and in vivo microenvironments. Due to the anti-apoptotic properties of melatonin, researchers have proposed that melatonin may improve the efficiency of spermatogonial stem cell (SSC) transplantation. However, the appropriate methodology which facilitates SSC proliferation remains to be determined. Identification of a proper propagation system is essential for the future application of SSCs in the field of infertility. The aim of the present study was to investigate the effects of melatonin on the colonization of SSCs. SSCs were isolated from the testes of three to six day old mice, and their purities were assessed by cytometry using Plzf antibody. Isolated testicular cells were cultured in the absence or presence of melatonin extract for two weeks. Suppression of differentiation and maintenance of spermatogonial stem cells was confirmed by alkaline phosphatase staining and immunocytochemistry using Plzf antibody. The number and diameter of the colonies of SSCs were assessed during the 7^th and 14^th days of culture, and the expression of Id4, Plzf, and C-kit were evaluated using real-time PCR at the end of the culture period. The survival rate of the cultured cells in the presence of melatonin was significantly higher than the control group. The number and diameter of colonies also increased in the cells treated with melatonin. The results of our study suggest that culture of SSCs with 100 μM melatonin supplementation can increase SSCs proliferation significantly.     

Effects of ionizing radiation on proliferation and differentiation of mouse induced pluripotent stem cells

The present study aimed to estimate the clonogenic and differentiation potential of induced pluripotent stem (iPS) cells exposed to ionizing radiation. Compared with mouse hematopoietic stem/progenitor cells, iPS cells were less sensitive to radiation. To examine the effect of ionizing radiation on the early differentiation pathway of iPS cells, we assessed embryoid body (EB) formation. Although EB formation was observed at all radiation doses, EB diameter decreased in a radiation dose-dependent manner. At the same time, we analyzed the expression of genes specific to differentiation in the initial iPS cells and cells of EB. The expression of the endoderm marker Afp increased remarkably in cells of EB derived from non-irradiated iPS cells; however, in irradiated cells, this expression significantly decreased in a radiation dose-dependent manner. Further, the expressions of the pluripotent stem cell markers Nanog and Oct-4 and the early mesoderm marker Brachyury significantly decreased. The results of the present study suggest that radiosensitivity with regard to gene expression differs at various stages in the early differentiation pathways of iPS cells that lead to the formation of the 3 germ layers; the sensitivity is the highest in the genes expressed during the differentiation pathways of iPS cells, leading to the formation of the endoderm.     

人肺腺癌肿瘤细胞免疫表型CDl33、CD34、CD44的实验研究

目的拟验证CDl33、CD34、CD44作为人肺腺癌肿瘤干细胞表面标记物的合理性。方法采集新鲜肺腺癌组织标本,利用两种体外培养方法扩增出贴壁细胞和悬浮细胞球两种肿瘤细胞,采用免疫荧光检测比较CDl33、CD34和CD44在两种培养细胞中表达的差异。结果悬浮球肿瘤细胞培养较贴壁肿瘤细胞生长速度慢、维持时间长且成功率高（72．5％VS47．5％,P〈0．05）。CDl33、CD34和CD44在悬浮细胞球中表达率和表达量明显高于贴壁肿瘤细胞（68．97％,82．76％,93．10％VS5．26％,15．79％,5．26％,P〈0．01）。结论CDl33、CD34和CD44可能作为分离人肺腺癌肿瘤干细胞的表面蛋白表标记组合。     

The mind-body connection: not just a theory anymore

The field of psychoneuroimmunology has witnessed an explosion of empirical findings during the last two decades. Research has documented the mechanisms through which stressful emotions alter white blood cell function. Stress diminishes white blood cell response to viral infected cells and to cancer cells. Moreover, vaccination is less effective in those who are stressed and wounds heal less readily in those who are stressed. While stress decreases the activity of some white blood cells, stress does not compromise the function of all types of white blood cells. Indeed, some types of autoimmune disease, which involve particular subsets of white blood cells, are exacerbated by stress. The literature documents the efficacy of talk-therapy interventions in altering immune system parameters and enhancing the body's ability to combat disease. The literature also documents the impact of the chronic stress of poverty on immune system function.