星形胶质细胞瘤干细胞的体外分离、培养与鉴定

背景：肿瘤干细胞理论认为肿瘤中存在一小部分具有无限增殖潜能和自我更新能力,能够分化为成熟细胞表型的干细胞样细胞,对肿瘤发生、增殖、侵袭起关键作用。 目的：建立体外分离、培养与鉴定星形胶质细胞瘤干细胞的方法。 方法：采用直接培养法分离培养星形胶质细胞瘤干细胞。参照神经干细胞培养条件,进行体外培养。观察其增殖、分化并进行巢蛋白、CD133免疫细胞化学鉴定和诱导分化后神经元特异性烯醇化酶、胶质纤维酸性蛋白及O4免疫细胞化学鉴定。 结果与结论：培养7-10 d,可形成大量悬浮生长巢蛋白及CD133免疫阳性的神经球,经诱导分化后细胞呈神经元特异性烯醇化酶、胶质纤维酸性蛋白或O4免疫阳性。提示星形胶质细胞瘤中存在具有神经干细胞特性的肿瘤干细胞。CD133和巢蛋白是星形胶质细胞瘤干细胞重要的表面标记,可以用于星形胶质细胞瘤干细胞的分离。     

Transplantation of Cultured Human Neural Progenitor Cells into Rat Brain: Migration and Differentiation

We studied the fate in vitro cultured human stem/progenitor cells after transplantation into rat brain. The cells from human fetuses at 8-12 weeks' gestation were cultured in vitro for 14 days and transplanted into the brain of 10-day-old and adult rats. Microscopic examination showed that human stem/progenitor cells migrated into various regions of rat brain. Immunohistochemical assay demonstrated that some cells differentiated into astrocytes and neurons, while others retained the embryonic phenotype.     

脐血间充质干细胞移植修复缺氧缺血损伤新生大鼠的脑功能

背景：课题计划从神经细胞替代、促进内源性神经干细胞增殖和分化、保护神经元、促进突触重建以及减轻脑白质损伤等方面来探讨脐血间充质干细胞系统移植对新生大鼠缺氧缺血脑损伤后神经功能的修复作用及其机制。 目的：观察脐血间充质干细胞由静脉途径移植透过血脑屏障进入脑组织内,对新生大鼠缺氧缺血性脑损伤后脑功能修复的影响。 方法：7 d龄SD新生鼠分为3组：假手术组仅分离出左侧颈总动脉而不结扎;缺氧缺血脑损伤组制备缺氧缺血脑损伤模型;细胞移植组在缺氧缺血性脑损伤后第8天尾静脉移植人脐血间充质干细胞,前两组尾静脉注射等量的生理盐水。 结果与结论：免疫荧光观察显示移植后5周脐血间充质干细胞迁移到海马,Nissl染色结果显示脐血间充质干细胞移植后,左侧海马DG区锥体细胞尼氏小体明显增加,提示间充质干细胞移植后可分化为神经元。行为学测试结果显示：与假手术组相比,缺氧缺血脑损伤组在T迷宫实验中,自发改变率下降,在放射形迷宫中觅水时间延长,错误次数及重复次数明显增加(P < 0.05);脐血间充质干细胞静脉移植5周后,上述行为学指标均显著改善(P < 0.05)。提示脐血间充质干细胞静脉移植治疗明显改善和提高了缺氧缺血脑损伤大鼠远期的学习记忆和空间辨别能力。     

人促红细胞生成素基因修饰人羊膜间充质干细胞蛛网膜下腔移植对脑损伤的影响

BACKGROUND:Studies have shown that as a regulator of bone marrow function erythropoietin is a glycoprotein that controls the development of the central nervous system and has neurotrophic and neuroprotective potential. Therefore, transplantation of human amniotic mesenchymal stem cells genetically modified by human erythropoietin is a new choice for brain injury treatment. OBJECTIVE:To observe the effect of transplantation of human amniotic mesenchymal stem cells genetically modified by human erythropoietin on the functional recovery from brain injury in rats. METHODS:Eukaryotic expression plasmid pcDNA3.1 carrying erythropoietin was successfully constructed and transferred into amniotic mesenchymal stem cells cultured in vitro. Expression of erythropoietin was detected using western blot assay before and after transfection. Rat models of middle cerebral arterial occlusion was made and given transplantation of transfected amniotic mesenchymal stem cells via the tail vein (transfection group). Additionally, model and simple cell transplantation groups were set in a comparative study. RESULTS AND CONCLUSION:Findings from western blot detection showed that transfected cells could express human erythropoietin. Compared with the other groups, modified neurologic severity scores, growth-associated protein 43 and aquaporin 9 at mRNA and protein levels were all decreased significantly in the transfection group. Furthermore, the number of cells positive for CM-Dil was highest in the transfection group, followed by simple cell transplantation group, and lowest in the model group (all P < 0.05). Overall findings from this study show that human erythropoietin-modified human amniotic mesenchymal stem cell transplantation promotes neurologic recovery from brain injury through eliciting a reduction in growth-associated protein 43 and aquaporin 9 at mRNA and protein levels as well as inhibiting cell apoptosis.     

Mechanisms of Positive Effects of Transplantation of Human Placental Mesenchymal Stem Cells on Recovery of Rats after Experimental Ischemic Stroke

Mesenchymal stem cells isolated from human placenta and in vitro labeled with fluorescent magnetic microparticles were intravenously injected to rats 2 days after induction of focal cerebral ischemia (endovascular model). According to MRT findings, transplantation of mesenchymal stem cells led to an appreciable reduction of the volume of ischemic focus in the brain. Two or three weeks after transplantation, labeled cells accumulated near and inside the ischemic focus, in the hippocampus, and in the subventricular zone of both hemispheres. Only few human mesenchymal stem cells populating the zone adjacent to the ischemic focus started expressing astroglial and neuronal markers. On the other hand, transplantation of mesenchymal stem cells stimulated proliferation of stem and progenitor cells in the subventricular zone and migration of these cells into the ischemic zone. Positive effects of transplantation of these cells to rats with experimental ischemic stroke are presumably explained by stimulation of proliferation of resident stem and progenitor cells of animal brain and their migration into the ischemic tissue and adjacent areas. Replacement of damaged rat neurons and glial cells by transplanted human cells, if it does take place, is quite negligible.     

Stem cell pathologies and neurological disease

The presence of stem and progenitor cells in the adult human brain suggests a putative and persistent role in reparative behaviors following neurological injury and neurological disease. Too few stem/progenitor cells (as in the case of Parkinson's disease) or too many of these cells (as in the case of Huntington's disease and glioma) could contribute to and even signal brain pathology. We address here critical issues faced by the field of stem cell biology and regenerative medicine, arguing from well-documented as well as speculative perspectives for a potential role for stem cells in the pathology of many human neurological diseases. Although stem cell responses may result in regenerative failure, in many cases they may help in the establishment or re-establishment of a functional neural circuitry (eg, after stroke). Therefore, we would argue that stem cells have a crucial-either positive or negative-role in the pathology of many neurological diseases.     

大脑皮质类器官培养及其在中枢神经系统疾病中的应用

文题释义： 大脑皮质类器官：利用三维细胞培养的方法,在合适的条件下将多能干细胞从单细胞生成一种能发育成神经上皮并具有类似活体大脑组织的复杂结构。 自我重组：像胚胎干细胞或诱导性多能干细胞这一类的多能干细胞,具有自发发育成某些原始结构的趋势。 背景：大脑组织是人体发育最复杂的结构,许多人类大脑疾病难以在动物体内重现,因此建立大脑发育的体外模型具有非常重要的研究价值。最近几年随着干细胞领域的飞速发展,出现了一种人工组织培养技术,即通过体外三维培养的方式,将干细胞从单个细胞生成一种复杂的、类似活体组织的结构,这种结构称为类器官。 目的：文章从大脑类器官的培养现状、发生机制、组织学特征及在神经系统疾病中的应用等多个方面综述了近年来大脑类器官的研究进展,并分析了目前大脑类器官的研究缺陷,旨在为相关领域的研究提供参考。 方法：由第一作者检索1998年1月至2019年6月中国知网、万方、PubMed数据库相关文献,检索词为“类器官、大脑类器官、胚胎干细胞、诱导性多能干细胞、神经发生、大脑皮质、发育、神经退行性疾病”“organoids,cerebral organoids,embryonic stem cells,induced pluripotent stem cells, neurogenesis,cerebral cortex,development,neurological diseases,self-organization”,纳入45篇文献进行综述。 结果与结论：利用多能干细胞自我重组的特性及添加神经诱导因子的方法,在体外可以有效产生大脑皮质类器官。作为一种全新的生物培养技术,大脑类器官在研究活体组织的发育、疾病形成的机制、组织替代疗法以及药物实验等方面都有很大的研究和应用价值。 ORCID: 0000-0002-8700-3030(范文娟) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Generation of Cancerous Neural Stem Cells Forming Glial Tumor by Oncogenic Stimulation

Neural stem cells in the brain have been shown to be 'cells of origin' of certain brain cancers, most notably astrocytomas and medulloblastoma. In particular, in a mouse model, the targeting of genetic modifications for astrocytoma-relevant tumor suppressors to neural stem cells causes malignant astrocytoma to arise, thereby suggesting that astrocytoma is derived from neural stem cells. However, it remains to be determined whether this important finding is reproducible in humans. Herein, we generated cancerous neural stem cells by introducing a set of oncogenes to human fetal neural stem cells (hfNSCs). Serial genetic modification with v-myc for immortalization and consequent H-Ras for oncogenic stimulation with viral gene delivery proved sufficient to induce the transformation of hfNSCs. The resultant F3.Ras cells evidenced a variety of the hallmarks of brain cancer stem cells and most importantly were tumorigenic, forming brain cancers consisting of both a large number of differentiated and a very few undifferentiated populations of cells in an in vivo mouse model. On the contrary, oligodendrocytes derived from the v-myc expressing parent neural stem cells were not transformed by H-Ras, which suggests that neural stem cells may be more susceptible to cancerous transformation by a combination of oncogenes. We also determined that v-myc expressing fetal neural stem cells were defective in p53 response upon the introduction of H-Ras; this finding suggests that an insufficient p53-dependent tumor suppressive mechanism would be associated with high oncogenic susceptibility to H-Ras introduction.     

人胚神经干细胞的分离、培养与鉴定

为了探讨人胚神经干细胞体外培养条件和分化情况，摸索出一种切实可行的获得较纯、多潜能人 胚神经干细胞的方法。我们取三月龄人胎脑，用胰蛋白酶消化法分离单个细胞，部分冻存，另一部分进行细胞培养，加EGF、bFGF刺激生长，有限稀释法将获得单细胞克隆，血清诱导分化，并用免疫组化方法进行鉴定。结果显示,EGF和bFGF同时存在的无血清培养基中有大量神经干细胞团生成，含血清培养基则诱导神经干细胞分化成为神经元、星型胶质细胞、少 突胶质细胞。这表明，神经干细胞的存活和分裂有赖于EGF和bEGF的共同作用。经冻存后的胎脑细胞同样能分离培养出有活性的神经干细胞。     

Brain cancer stem cells display preferential sensitivity to Akt inhibition

Malignant brain tumors are among the most lethal cancers, and conventional therapies are largely limited to palliation. Novel therapies targeted against specific molecular pathways may offer superior efficacy and less toxicity than conventional therapies, but initial clinical trials of molecular targeted agents in brain cancer therapy have been frequently disappointing. In brain tumors and other cancers, subpopulations of tumor cells have recently been characterized by their ability to self-renew and initiate tumors. Although these cancer stem cells, or tumor initiating cells, are often only present in small numbers in human tumors, mounting evidence suggests that cancer stem cells contribute to tumor maintenance and therapeutic resistance. Thus, the development of therapies that target cancer stem cell signal transduction and biology may improve brain tumor patient survival. We now demonstrate that populations enriched for cancer stem cells are preferentially sensitive to an inhibitor of Akt, a prominent cell survival and invasion signaling node. Treatment with an Akt inhibitor more potently reduced the numbers of viable brain cancer stem cells relative to matched nonstem cancer cells associated with a preferential induction of apoptosis and a suppression of neurosphere formation. Akt inhibition also reduced the motility and invasiveness of all tumor cells but had a greater impact on cancer stem cell behaviors. Furthermore, inhibition of Akt activity in cancer stem cells increased the survival of immunocompromised mice bearing human glioma xenografts in vivo. Together, these results suggest that Akt inhibitors may function as effective anticancer stem cell therapies.     

免疫磁珠体外纯化人胎脑中CD133阳性干细胞的实验研究

为了探讨免疫磁珠体外纯化人胎儿脑内 CD13 3阳性细胞的可行性 ,本研究取人胎儿脑室下区并制备单细胞悬液 ,采用磁珠分选法选择 CD13 3阳性细胞群 ,台盼蓝染色观察活力并采用流式细胞仪鉴定纯化前后 CD13 3的阳性表达率。结果显示 ,分选后所得细胞纯度为 ( 85 .5 7± 1.66) % ,回收率为 ( 62 .3± 18) % ,纯化前后细胞活力无显著性差异 ( P>0 .0 5 )。结论 :联合应用CD13 3表面标志及免疫磁珠分选系统可有效地从人胎儿脑组织中直接分离得到高纯度的 CD13 3阳性干细胞 ,细胞活力不受影响     

Human neural stem cells can migrate,differentiate, and integrate after intravenous transplantation in adult rats with transient forebrain ischemia

Intraparenchymally transplanted rodent-origin neural and human-origin mesenchymal stem cells migrate and differentiate in neurological diseases. By intravenously injecting human neural stem cells, we showed that transplanted human neural stem cells migrate to the damaged hippocampus, proliferate and differentiate into mature neurons and astrocytes in the adult rat brain with transient forebrain ischemia. We also demonstrated the migratory course of implanted human neural stem cells after intravenous injection. Our findings show that transplanted human neural stem cells differentiate into mature neurons to replace lost neural cells in the adult hippocampus with human-rat neural chimeras.     

Noggin enhances dopamine neuron production from human embryonic stem cells and improves behavioral outcome after transplantation into Parkinsonian rats

Symptoms of Parkinson's disease have been improved by transplantation of fetal dopamine neurons recovered from aborted fetal tissue, but tissue recovery is difficult. Human embryonic stem cells may provide unlimited cells for transplantation if they can be converted to dopamine neurons and survive transplantation into brain. We have found that the bone morphogenic protein antagonist Noggin increased the number of dopamine neurons generated in vitro from human and mouse embryonic stem cells differentiated on mouse PA6 stromal cells. Noggin effects were seen with either early (for mouse, days 0-7, and for human, days 0-9) or continuous treatment. After transplant into cyclosporin-immunosuppressed rats, human dopamine neurons improved apomorphine circling in direct relation to the number of surviving dopamine neurons, which was fivefold greater after Noggin treatment than with control human embryonic stem cell transplants differentiated only on PA6 cells. We conclude that Noggin promotes dopamine neuron differentiation and survival from human and mouse embryonic stem cells. Disclosure of potential conflicts of interest is found at the end of this article.     

Spontaneous transformation of human adult nontumorigenic stem cells to cancer stem cells is driven by genomic instability in a human model of glioblastoma

The presence of a CD133+/nestin+ population in brain tumors suggests that a normal neural stem cell may be the cell of origin for gliomas. We have identified human CD133-positive NSCs from adult glioma tissue and established them as long-term in vitro cultures human neuroglial culture (HNGC)-1. Replicative senescence in HNGC-1 led to a high level of genomic instability and emergence of a spontaneously immortalized clone that developed into cell line HNGC-2 with features of cancer stem cells (CSCs), which include the ability for self-renewal and the capacity to form CD133-positive neurospheres and develop intracranial tumors. The data from our study specify an important role of genomic instability in initiation of transformed state as well as its progression into highly tumorigenic CSCs. The activated forms of Notch and Hes isoforms were expressed in both non-neoplastic neural stem cells and brain tumor stem cells derived from it. Importantly, a significant overexpression of these molecules was found in the brain tumor stem cells. These findings suggest that this model comprised of HNGC-1 and HNGC-2 cells would be a useful system for studying pathways involved in self-renewal of stem cells and their transformation to cancer stem cells. Disclosure of potential conflicts of interest is found at the end of this article.     

New drugs for brain tumors? Insights from chemical probing of neural stem cells

The cancer stem cell hypothesis posits a direct relationship between normal neural stem cells (NSCs) and brain tumour stem cells (BTSCs). New insights into human brain tumour biology and treatment should thus emerge from the study of normal NSCs. These parallels have recently been exploited in a chemical genetic screen that identified a broad repertoire of neurotransmission modulators as inhibitors of both NSC and BTSC expansion in vitro. Prompted by these findings, we sought epidemiological support for effects of neuromodulation of brain tumours in vivo. We present observations from data collected from retrospective clinical studies suggesting that patients with a wide variety of neuropsychiatric disorders have decreased brain tumour incidence. We speculate that this reduction may derive from the use of drugs that collaterally affect the normal neural precursor compartment, and thereby limit a population that is suspected to give rise to brain tumours. Standard chronic neuropharmacological interventions in clinical neuropsychiatric care are thus candidates for redeployment as brain cancer therapeutics.     

Immunohistochemical Study of Fetal Stem/Progenitor Cells from Human Brain Transplanted into Traumatized Spinal Cord of Adult Rats

Neural stem/progenitor cells from human fetal brain were grown in a tissue culture and transplanted into traumatized spinal cord of adult rats. The behavior and differentiation of transplanted cells were studied morphologically by means of histological and immunohistochemical methods and confocal microscopy. Human neural stem/progenitor cells were viable for not less than 3 months. They migrated and differentiated into neurons and glia in the traumatized spinal cord of adult rats.     

Neuroprotective Effect of Mesenchymal and Neural Stem and Progenitor Cells on Sensorimotor Recovery after Brain Injury

We studied the effect of systemic administration of multipotent stem cells on impaired neurological status in rats with brain injury. It was found that transplantation of multipotent mesenchymal stromal cells of the bone marrow or human neural stem and progenitor cells to rats with local brain injury promoted recovery of the brain control over locomotor function and proprioceptive sensitivity of forelegs. The dynamics of neurological recovery was similar after transplantation of fetal neural stem and progenitor cells and multipotent mesenchymal stromal cells. Transplantation of cell cultures improved survival of experimental animals. It should be noted that administration of neural stem and progenitor cells prevented animal death not only in the acute traumatic period, but also in delayed periods.     

Comparative analysis of differentiation and behavior of human neural and mesenchymal stem cells In Vitro and In Vivo

Comparative analysis of differentiation of human neural and mesenchymal stem cells in tissue culture and after transplantation into the brain was carried out using the same antibody set. Neural stem cells differentiated into all types of neural cells, are retained after transplantation, migrate, and form reciprocal relationships with the recipient brain. Mesenchymal stem cells were incapable of neural development under conditions of common culturing or after transplantation and retained the fibroblast-like status. Recipient filaments grew into mesenchymal stem cell transplants containing no neural cells due to local changes in the extracellular matrix at the site of transplantation. __________ Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 44–52, January, 2006