氯甲基苯甲酰氨荧光染料标记大鼠骨髓间质干细胞的体内示踪

背景：目前广泛应用于骨髓间质干细胞的标记方法主要为绿色荧光蛋白标记法，但标记与固定程序复杂，操作繁琐，易出现偏差。氯甲基苯甲酰氨(Chloromethyl-benzamidodialkylcarbocyanine, CM-Dil)是亲脂性膜荧光染料，能够与含有肽和蛋白质的硫氢基结合进而标记整个细胞。 目的：探讨CM-Dil对大鼠骨髓间质干细胞体内示踪的可行性。 设计、时间及地点：体内外细胞学观察，于2007-05/12在中山大学干细胞与组织工程研究中心、中山大学动物实验中心完成。 材料：SPF级Wistar大鼠40只，由中山大学实验动物中心提供。CM-Dil为美国Sigma公司产品。 方法：在体外，以标记CM-Dil的骨髓间质干细胞作为实验组，以未标记CM-Dil的骨髓间质干细胞作为对照组，比较两组细胞生长与增殖情况，MTT法检测细胞生长增殖情况。在体内，分别将标记CM-Dil的骨髓间质干细胞经门静脉移植及肝内培养，于移植后第7，15，21，30天制备肝脏切片。 主要观察指标：骨髓间质干细胞CM-Dil标记率，标记细胞在肝内定植、生长与分布。 结果：体外培养结果显示，两组骨髓间质干细胞在细胞生长、增殖、分裂以及形态学上均基本相似，在绿色光激发下，CM-Dil发出红色荧光，24 h后细胞标记率为100%，21 d后CM-Dil标记的骨髓间质干细胞荧光开始淬灭。体内实验中，经门静脉移植的骨髓间质干细胞在肝脏内主要位于间质，呈椭圆形或不规则形的分化细胞；肝内培养的骨髓间质干细胞在培养孔内呈“贴壁生长”，为椭圆形分化细胞，未发现骨髓间质干细胞进入并定植于肝组织内；CM-Dil标记的骨髓间质干细胞荧光开始淬灭的时间亦为21 d。 结论：CM-Dil染色简单、方便、稳定，荧光开始淬灭时间较长，可望成为大鼠骨髓间质干细胞体内示踪的新方法。     

Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axons.

The potential of bone marrow cells to differentiate into myelin-forming cells and to repair the demyelinated rat spinal cord in vivo was studied using cell transplantation techniques. The dorsal funiculus of the spinal cord was demyelinated by x-irradiation treatment, followed by microinjection of ethidium bromide. Suspensions of a bone marrow cell fraction acutely isolated from femoral bones in LacZ transgenic mice were prepared by centrifugation on a density gradient (Ficoll-Paque) to remove erythrocytes, platelets, and debris. The isolated cell fraction contained hematopoietic and nonhematopoietic stem and precursor cells and lymphocytes. The cells were transplanted into the demyelinated dorsal column lesions of immunosuppressed rats. An intense blue beta-galactosidase reaction was observed in the transplantation zone. The genetically labeled bone marrow cells remyelinated the spinal cord with predominately a peripheral pattern of myelination reminiscent of Schwann cell myelination. Transplantation of CD34(+) hematopoietic stem cells survived in the lesion, but did not form myelin. These results indicate that bone marrow cells can differentiate in vivo into myelin-forming cells and repair demyelinated CNS.     

Isolation and differentiation of embryonic stem cells from BALB/c mouse

Objective To invest the efficient method which can culture and induce embryonic stem cells to neurocyte in vitro. Methods Isolate the blastula of 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass (inner cell mass, ICM) which were isolated by mechanical method on the mouse embryonic fibroblaste cell (MEF) feeder layer or 0.1% gelatin coated dishes. The stem cells were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunochemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 (stage specific embryonic antigen 1). Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated cells presented the characters of ESCs. Then the isolated cells were able to differentiate into neurocytes in vitro. Conclusion Mouse embryonic stem cells isolation, culture and differentiation system has been established.     

Acute transplantation of glial-restricted precursor cells into spinal cord contusion injuries: survival, differentiation, and effects on lesion environment and axonal regeneration

Transplantation of stem cells and immature cells has been reported to ameliorate tissue damage, induce axonal regeneration, and improve locomotion following spinal cord injury. However, unless these cells are pushed down a neuronal lineage, the majority of cells become glia, suggesting that the alterations observed may be potentially glially mediated. Transplantation of glial-restricted precursor (GRP) cells--a precursor cell population restricted to oligodendrocyte and astrocyte lineages--offers a novel way to examine the effects of glial cells on injury processes and repair. This study examines the survival and differentiation of GRP cells, and their ability to modulate the development of the lesion when transplanted immediately after a moderate contusion injury of the rat spinal cord. GRP cells isolated from a transgenic rat that ubiquitously expresses heat-stable human placental alkaline phosphatase (PLAP) were used to unambiguously detect transplanted GRP cells. Following transplantation, some GRP cells differentiated into oligodendrocytes and astrocytes, retaining their differentiation potential after injury. Transplanted GRP cells altered the lesion environment, reducing astrocytic scarring and the expression of inhibitory proteoglycans. Transplanted GRP cells did not induce long-distance regeneration from corticospinal tract (CST) and raphe-spinal axons when compared to control animals. However, GRP cell transplants did alter the morphology of CST axons toward that of growth cones, and CST fibers were found within GRP cell transplants, suggesting that GRP cells may be able to support axonal growth in vivo after injury.     

Direct isolation of committed neuronal progenitor cells from transgenic mice coexpressing spectrally distinct fluorescent proteins regulated by stage-specific neural promoters

Many tissues arise from pluripotent stem cells through cell-type specification and maturation. In the bone marrow, primitive stem cells generate all the different types of blood cells via the sequential differentiation of increasingly committed progenitor cells. Cell-surface markers that clearly distinguish stem cells, restricted progenitors, and differentiated progeny have enabled researchers to isolate these cells and to study the regulatory mechanisms of hematopoiesis. Neuronal differentiation appears to involve similar mechanisms. However, neural progenitor cells that are restricted to a neuronal fate have not been characterized in vivo, because specific cell-surface markers are not available. We have developed an alternative strategy to identify and isolate neuronal progenitor cells based on dual-color fluorescent proteins. To identify and isolate directly progenitor cells from brain tissue without the need for either transfection or intervening cell culture, we established lines of transgenic mice bearing fluorescent transgenes regulated by neural promoters. One set of transgenic lines expressed enhanced yellow fluorescent protein (EYFP) in neuronal progenitor cells and neurons under the control of the Talpha1 alpha-tubulin promoter. Another line expressed enhanced green fluorescent protein (EGFP) in immature neural cells under the control of the enhancer/promoter elements of the nestin gene. By crossing these lines we obtained mice expressing both transgenes. To isolate neuronal progenitor cells directly from the developing brain, we used flow cytometry, selecting cells that expressed EGFP and EYFP simultaneously. We expect this strategy to provide valuable material with which to study the mechanisms of neurogenesis and to develop cell-based therapies for neurological disorders.     

Clonal neural stem cells from human embryonic stem cell colonies

Clonal cell culture is crucial for experimental protocols that require growth or selection of pure populations of cells. High-density derivation of neural progenitors from human embryonic stem cells (hESCs) can lead to incomplete differentiation, and transplantation of resulting heterogeneous cell mixtures can cause proliferation of tumorigenic clusters in vivo. We have identified the neural precursor that resides among normal hESC colonies as a TRA-1-60(-)/SSEA4(-)/SOX1(+) cell and developed a method that allows for the clonal expansion of these FACS-selected progenitors to neural stem cells (NSCs) in serum-free conditions. Single TRA-1-60(-)/SSEA4(-)/SOX1(+) cells grown in serum-free media give rise to multipotent NSCs with an efficiency of 0.7%. The fate of the TRA-1-60(-)/SSEA4(-)/SOX1(+) neural precursor becomes specified in maintenance conditions by inhibition of BMP signaling. This clonal culture method can be scaled up to produce NSCs for differentiation and use in cell therapies.     

Characterization of long-term mouse brain aggregating cultures: evidence for maintenance of neural precursor cells

An extensive characterization of fetal mouse brain cell aggregates has been performed using immunohistochemical and stereological methods. Single cell suspensions from mechanically dissociated cortex and hippocampus were cultured in serum-free, B27-supplemented medium under constant gyratory agitation for up to 56 days. Three-dimensional aggregates started to form immediately after seeding and reached a final average size of 500 microm in diameter. Among the cell types identified, neurons were the most abundant cells in the aggregates, followed by astrocytes, microglia, and oligodendrocytes. Western blotting for synaptophysin and immunostaining for neurotransmitter-related molecules indicated the presence of well-defined phenotypic characteristics of the neurons in this culture system, suggesting functionality. Proliferating cells, many with neural precursor cell properties, were seen throughout the culture period and could be isolated from the aggregates even after 2 months in culture. Neural precursor cells were isolated from the aggregates after more than 1 month in culture; these cells were successfully differentiated into neurons, astrocytes, and oligodendrocytes. The aggregate culture system may provide a versatile tool for molecular dissection of processes identified in mouse models, including transgenic animals and manipulation of neural precursor cells.     

Loss of gene expression in lentivirus- and retrovirus-transduced neural progenitor cells is correlated to migration and differentiation in the adult spinal cord

Gene transfer into multipotent neural progenitor cells (NPC) and stem cells may provide for a cell replacement therapy and allow the delivery of therapeutic proteins into the degenerating or injured nervous system. Previously, murine leukemia virus-based retroviral vectors expressing GFP from an internal EF-1alpha promoter and lentiviral vectors expressing GFP from a hybrid CMV/beta-actin promoter have been described to be resistant to stem cell specific gene silencing. Therefore, we investigated whether these viral vectors allow stable in vivo gene expression in genetically modified NPC isolated from the adult rat spinal cord. In vitro, NPC genetically modified to express GFP using the described retroviral vector showed strong GFP expression in undifferentiated NPC. However, in vitro differentiation resulted in the loss of GFP expression in 50% of cells. Grafting of BrdU-prelabeled NPC to the spinal cord resulted in a loss of GFP expression in 70% and 95% of surviving NPC at 7 and 28 days post-grafting, respectively. The loss in gene expression was paralleled by the differentiation of NPC into a glial phenotype. Transgene downregulation although less profound was also observed in cells modified with lentiviral vectors, whereas in vivo lentiviral gene transfer resulted in stable transgene expression for up to 16 months. Thus, in vivo gene expression in genetically engineered neural progenitor cells is temporally limited and mostly restricted to undifferentiated NPC using the viral vectors tested.     

大鼠中脑神经前体细胞的分离鉴定和培养

目的确认从E14.5SD大鼠中脑胚胎分离的细胞符合神经前体细胞特性,并在体外建立合适培养体系使神经前体细胞能够长期生长及传代。方法分离E14.5SD大鼠胚胎中脑腹侧组织细胞,在体外含有碱性纤维细胞生长因子(bFGF)的无血清培养液内种植并传代,行nestin免疫学检查,并在分化前后行神经元特异性烯醇化酶(NSE)和胶质原纤维酸性蛋白(GFAP)免疫学检查,同时做BrdU增殖实验。结果体外种植中脑神经细胞可以生长、分裂并长期传代,nestin染色及BrdU增殖实验为阳性,NSE和GFAP 在分化前阴性,而分化后为阳性。结论E14.5SD大鼠胚胎中脑分离的细胞符合神经前体细胞特性,并可在本实验所采用的培养液内长期生长、分裂和传代。     

Processing of the beta-amyloid precursor protein in ex vivo human brain cells

We studied distribution and processing of the Alzheimer's beta-amyloid precursor protein (betaAPP) in immediately ex vivo human brain cells obtained during neurosurgical procedures. Immunoblotting and flow cytometry studies revealed that brain cells supported betaAPP as a transmembrane holoprotein. Brain cells in short-term suspension culture were competent to process betaAPP into Abeta as shown by [35S]methionine pulse-chase studies. Brain cell Abeta was immunoprecipitated as SDS-stable dimers and higher-order multimers. Cleavage of cell surface betaAPP with trypsin prior to metabolic labeling reduced cellular Abeta by approximately 50%. We conclude that plasmalemmal betaAPP in human brain cells is a source of cellular Abeta, presumably via endosomal-lysosomal processing.     

Human muscle precursor cells give rise to functional satellite cells in vivo

Mouse satellite cells have been shown to be functional muscle stem cells, in that they are able to regenerate skeletal muscle and to reconstitute the satellite cell pool. Although human muscle precursor cells are able to contribute to skeletal muscle regeneration following transplantation into host mouse muscles, it is uncertain whether they also give rise to functional satellite cells. Here, we transplant human fetal muscle precursor cells into cryodamaged muscles in C(5)-/gamma-chain-/Rag2-host mice. The donor cells gave rise to muscle fibres that persisted for up to 6 months after grafting. Isolated muscle fibres, bearing satellite cells, were prepared from muscles 4 weeks after grafting. When placed in culture, a small proportion of these fibres gave rise to muscle precursor cells of human origin, indicating that the originally grafted cells had formed satellite cells as well as regenerated muscle fibres. These satellite cell-derived human muscle precursor cells were expanded in culture and formed muscle following their transplantation into a second series of host mice. This provides evidence that human, as well as mouse, muscle precursor cells, are capable of forming functional satellite cells in vivo.     

人神经干细胞的分离、克隆和动物脑内移植及转基因表达

目的分离和克隆人神经干细胞,并在体外和体内分析其生物学特征.方法我们联合采用四步法从人胚胎前脑分离制备多潜能神经干细胞,并使用重组腺病毒相关病毒载体(rAAV)将LacZ基因和胶质细胞起源的神经营养因子(GDNF)基因转移到神经干细胞.结果二株人神经干细胞被成功建立.这些克隆化后的神经干细胞在细胞培养中和移植到新生小鼠脑内后能发育分化成神经元、少枝胶质细胞和星形胶质细胞.在rAAV转导基因后,神经干细胞可在体外和体内表达转基因产物.结论这种具有转基因表达能力的神经干细胞为神经系统疾病的进一步治疗研究提供了有潜在价值的细胞资源.     

Isolation and differentiation of embryonic stem cells from BALB/c mouse

Objective To invest the efficient method which can culture and induce embryonic stem cells to neuroeyte in vitro. Methods Isolate the blastula o f 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass （inner cell mass, ICM） which were isolated by mechanical method on the mouse embryonic fibroblaste cell （MEF） feeder layer or 0.1% gelatin coated dishes. The stem ceils were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunoehemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 （ stage specific embryonic antigen 1 ）. Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated ceils presented the characters of ESCs. Then the isolated cells were able to differentiate into neuroeytes in vitro. Conclusion Mouse embryonic stem ceils isolation, culture and differentiation system has been established.     

Absence of hematopoiesis from transplanted olfactory bulb neural stem cells

Neural stem cells giving rise to neurons and glia cells have been isolated from the embryonic and adult central nervous system. The extent to which they are able to differentiate into cells of non-neural lineages, such as the hematopoietic lineage, is nonetheless unclear. We previously reported the isolation of stem cells from the mouse olfactory bulb neuroepithelium. In the present study, we analysed whether olfactory bulb stem cells (OBSC) can generate cells with hematopoietic features. Cells were prepared from the olfactory bulbs of transgenic mice expressing enhanced green fluorescent protein (EGFP). In culture, transgenic cells proliferated with the same kinetics as wild-type cells. Following mitogen removal, both cell types gave rise to similar numbers of neurons, astrocytes and oligodendrocytes, indicating that EGFP overexpression does not alter OBSC proliferation and differentiation patterns. When these cells were injected into the tail vein of irradiated mice, no hematopoietic cells derived from the OBSC could be recovered in their peripheral blood, spleen or bone marrow. By contrast, when OBSC were transplanted into the adult brain, EGFP-positive cells were found in the striatum and corpus callosum; differentiated cells expressed antigenic markers of neurons and astrocytes. These results suggest that embryonic olfactory bulb stem cells are not endowed with the potential to produce hematopoiesis.     

Human stem cells isolated from adult skeletal muscle differentiate into neural phenotypes

Multipotent neural stem cells have been isolated from the adult [Kirschenbaum B, Nedergaard M, Preuss A, Barami K, Fraser RA, Goldman SA. In vitro neuronal production and differentiation by precursor cells derived from the adult human forebrain. Cereb Cortex 1994;4(6):576-89; Laywell ED, Kukekov VG, Steindler DA. Multipotent neurospheres can be derived from forebrain subependymal zone and spinal cord of adult mice after protracted postmortem intervals. Exp Neurol 1999;156:430-3; Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani G, Dina G, et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 2003;422:688-94] and embryonic [Vescovi AL, Parati EA, Gritti A, Poulin P, Ferrario M, Wanke E, et al. Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp Neurol 1999;156:71-83] central nervous system (CNS). In addition, neural cells can be obtained from sources other than the CNS by differentiating stem cells from a non-neural source down a neural lineage. This has previously been performed with pluripotent embryonic stem cells and adult stem cells derived from rat bone marrow [Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000;61:364-70; Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci 2002;69(6):908-17] and skeletal muscle [Romero-Ramos M, Vourc'h P, Young HE, Lucas PA, Wu Y, Chivatakarn O, et al. Neuronal differentiation of stem cells isolated from adult muscle. J Neurosci Res 2002;69:894-907]. Previously, we have isolated adult stem cells from human skeletal muscle with the potential to differentiate into mesoderm, ectoderm, and endoderm. The following in vitro experiments were designed to determine whether human adult stem cells behaved similarly to rat adult stem cells when both were isolated from skeletal muscle by the same procedure [Romero-Ramos M, Vourc'h P, Young HE, Lucas PA, Wu Y, Chivatakarn O, et al. Neuronal differentiation of stem cells isolated from adult muscle. J Neurosci Res 2002;69:894-907] and subjected to the same protocols to induce neurogenesis. The neural phenotypes that were created through the neurococktail or neurosphere protocol were analyzed for neural characteristics through morphology and immunohistochemistry antibody labeling for proteins to neurons (RT-97, beta-tubulin III, NF-160, NF-200, and synapsin), oligodendrocytes (CNPase and RIP), and astrocytes (GFAP). A calcium uptake assay also showed response to the neuronal excitotoxic agent glutamic acid. In conclusion, the neural differentiated stem cells derived from adult skeletal muscle may be a less invasive alternative for the treatment of CNS disorders over CNS derived neural stem cells.     

Differential gene expression in neural stem cells and oligodendrocyte precursor cells: a cDNA microarray analysis

The use of neural stem cells (NSCs) or their progeny oligodendrocyte precursor cells (OPCs) represents a promising repair strategy for many neurological disorders. However, the molecular events and biological features during the transition from NSCs to OPCs remain unclear. In the present study, we isolated NSCs from the embryonic rat forebrain and induced them into OPCs by using B104 conditioned medium (B104CM) in vitro. We then employed cDNA array technology to compare changes in gene expression between the two cell populations. Among 1,176 genes examined, 40 were differentially expressed, and some of them may be involved in OPC differentiation from NSCs. Our findings thus provide new insights into the molecular basis of differentiation of OPCs from NSCs.     

胶质瘤干细胞中Nanog基因启动子区甲基化检测及意义

目的检测胶质瘤干细胞中Nanog基因启动子区甲基化表达,并探讨其Nanog基因表达的关系。方法使用无血清悬浮培养法获得胶质瘤干细胞并鉴定;采用甲基化特异性多聚酶链反应(MSP)分别检测胶质瘤干细胞和胶质瘤细胞系U87中Nanog基因启动子区甲基化状态,实时定量多酶链反应(RT-PCR)检测相应细胞中Nanog表达情况。结果由U87胶质瘤细胞系成功获得胶质瘤干细胞(GSCs);MSP检测胶质瘤干细胞和U87细胞系Nanog启动子区皆呈非甲基化状态,且胶质瘤干细胞中Nanog非甲基化程度高于U87细胞系(t=6.988,P=0.000)。实时定量PCR结果显示胶质瘤干细胞中Nanog mRNA相对表达量高于U87细胞系。结论胶质瘤干细胞中Nanog基因启动子区呈非甲基化状态,可能与Nanog基因表达有关,且可能促进Nanog的转录并维持着胶质瘤干细胞的恶性生物学活性。