Practical issues in stem cell therapy for Alzheimer's disease

We have demonstrated that aged animals show significant improvements in cognitive function and neurogenesis after brain transplantation of human neural stem cells or of human adult mesenchymal stem cells that have been dedifferentiated by transfection of the embryonic stem cell gene. We have also demonstrated that peripheral administration of a pyrimidine derivative increased cognition, endogenous brain stem cell proliferation and neurogenesis. These results indicate a bright future for stem cell therapies in Alzheimer's disease (AD). Before this is realized, however, we need to consider the affect of AD pathology on stem cell biology to establish an effective stem cell therapy for this disease. Although amyloid-beta (Abeta) deposition is a hallmark of AD, an absence of a phenotype in the beta-amyloid precursor protein (APP) knockout mouse, might lead one to underestimate the potential physiological functions of APP and suggest that it is unessential or can be compensated for. We have found, however, that APP is needed for differentiation of neural stem cells (NSCs) in vitro, and that NSCs transplanted into a APP-knockout mouse did not migrate or differentiate -- indicating that APP plays an important role in differentiation or migration process of NSCs in the brain. Then again, treatment with high a concentration of APP or its over-expression increased glial differentiation of NSCs. Human NSCs transplanted into APP-transgenic mouse brain exhibited less neurogenesis and active gliosis around the plaque like formations. Treatment of such animals with the compound, (+)-phenserine, that is known to reduce APP protein levels, increased neurogenesis and suppressed gliosis. These results suggest APP levels can regulate NSC biology in the adult brain, that altered APP metabolism in Down syndrome or AD may have implications for the pathophysiology of these diseases, and that a combination of stem cell therapy and regulation of APP levels could provide a treatment strategy for these 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.     

Adult stem cell therapy for acute brain injury in children

Adult stem cell therapy has been proposed for brain injury in young children. While there have been no clinical trials in the US, the therapy is widely advertised and anecdotally reported in multiple internet sources, leading families to seek the treatment in uncontrolled circumstances. The purpose of this review is to present a discussion of the various types of stem cell preparations, with emphasis on adult stem cells, the scientific basis of their development, and the available experimental evidence for their utility in childhood brain injury. We will also provide background information on the biologic events occurring in injured immature brain, as they relate to the transplantation of stem cells. We will then review our own data from neonatal rodent studies with experimental hypoxic-ischemic brain injury. We have shown that early intracerebral administration promotes improved behavioral outcome in the animals, the formation of new neurons, and the preservation of intrinsic cells. New experiments demonstrate the equality of intracerebral and intravenous transplantation in acute neonatal hypoxic-ischemic injury in rodent. We will speculate on the possible clinical uses of adult stem cells. Our current impression is that the cells have the greatest potential for success when administered soon after an injury. What needs to be done to further the field? The different types of cell preparations should be tested against each other in experimental situations. A suitable model of chronic brain injury should be utilized for evaluating the benefit of the cells for this purpose. Long term safety of the cells should be confirmed in animal models. Finally, multicenter clinical trials should be conducted in highly controlled protocols.     

Genetically engineered human neural stem cells for brain repair in neurological diseases

Neural stem cells (NSCs)of the central nervous system (CNS) have recently received a great deal of attention and interest for their therapeutic potential for neurological disorders. NSCs are defined as CNS progenitor cells that have the capacity for self-renewal and multipotent potential to become neurons or glial cells. Recent studies have shown that NSCs isolated from mammalian CNS including human can be propagated in vitro and then implanted into the brain of animal models of human neurological disorders. Recently, we have generated clonally derived immortalized human NSC cell lines via a retroviral vector encoded with v-myc oncogene. One of the human NSC lines, HB1.F3, was utilized in stem-cell based therapy in animal models of human neurological disorders. When F3 human NSCs were implanted into the brain of murine models of lysosomal storage diseases, stroke, Parkinson disease, Huntington disease or stroke, implanted F3 NSCs were found to migrate to the lesion sites, differentiate into neurons and glial cells, and restore functional deficits found in these neurological disorders. In animal models of brain tumors, F3 NSCs could deliver a bioactive therapeutically relevant molecules to effect a significant anti-tumor response intracranial tumor mass. Since these genetically engineered human NSCs are immortalized and continuously multiplying, there would be limitless supply of human neurons for treatment for patients suffering from neurological disorders including stroke, Parkinson disease, Huntington disease, ALS, multiple sclerosis and spinal cord injury. The promising field of stem cell research as it applies to regenerative medicine is still in infancy, but its potential appears limitless, and we are blessed to be involved in this exciting realm of research.     

利用冷冻胚胎建立人类胚胎干细胞系

背景：目前人胚胎干细胞建系的技术路线主要有囊胚法、核移植法和胚胎单卵裂球法,但由于破坏胚胎,涉及诸多伦理问题。 目的：探讨利用冷冻胚胎建立人类胚胎干细胞系的可行性。 设计、时间及地点：细胞学体内外实验,于2005-01/2006-08在沈阳市妇婴医院生殖中心完成。 材料：妊娠13.5 d的ICR鼠20只用于制备小鼠胚胎成纤维细胞饲养层,10周龄SICD鼠2只用于人胚胎干细胞体内分化实验。行IVF/ICSI助孕患者自愿捐献的冷冻胚胎,由沈阳市妇婴医院生殖中心提供。 方法：复苏冷冻的胚胎,采用序贯培养法进行囊胚培养,用免疫外科方法去除滋养细胞,将得到的胚胎内细胞团接种于丝裂霉素C灭活的小鼠胚胎成纤维细胞上培养并传代。 主要观察指标：取不同代次的培养细胞,分别进行生长特性、碱性磷酸酶染色、转录因子OCT-4、阶段特异性胚胎抗原SSEA-4、SSEA-1、肿瘤排斥抗原TRA-1-60、TAR-1-81、核型及体内分化全能性鉴定。 结果：20个解冻卵裂期胚胎,获得9个囊胚,分离完整的内细胞团共7个,从7个内细胞团培养出2个人胚胎干细胞系,其中一个细胞系连续培养76代(20个月)。所培养的细胞具有人胚胎干细胞的共同生物学特性,即细胞呈扁平或圆形,可见1~3个核仁;碱性磷酸酶以及SSEA-3,SSEA-4,TRA-1-81,TRA-1-60,OCT-4均呈阳性表达,而SSEA-1呈阴性;核型正常,为46,XX核型;将细胞注射入SCID鼠皮下形成肿瘤,病理切片显示为成熟畸胎瘤;短串联重复分型结果显示所形成的畸胎瘤与人胚胎干细胞为相同的遗传背景。 结论：实施体外受精-胚胎移植的夫妇怀孕后,多余的冷冻保存胚胎所分离培养的细胞具备人胚胎干细胞的所有特性,是建立人胚胎干细胞系很好的材料来源。     

Engineered adenosine-releasing cells for epilepsy therapy: Human mesenchymal stem cells and human embryonic stem cells

Adenosine is a modulator of neuronal activity with anticonvulsant and neuroprotective properties. Conversely, focal deficiency in adenosine contributes to ictogenesis. Thus, focal reconstitution of adenosine within an epileptogenic brain region constitutes a rational therapeutic approach, whereas systemic augmentation of adenosine is precluded by side effects. To meet the therapeutic goal of focal adenosine augmentation, genetic disruption of the adenosine metabolizing enzyme, adenosine kinase (ADK) in rodent cells was used as a molecular strategy to induce adenosine release from cellular brain implants, which demonstrated antiepileptic and neuroprotective properties. Currently, the second generation of adenosine-releasing cells is under development based on the rationale to use human stem cell-derived brain implants to avoid xenotransplantation. To effectively engineer human stem cells to release adenosine, a lentiviral vector was constructed to express inhibitory micro-RNA directed against ADK. Lentiviral knockdown of ADK induced therapeutic adenosine release in human mesenchymal stem cells, which reduced acute injury and seizures, as well as chronic seizures, when grafted into the mouse hippocampus. The therapeutic potential of this approach suggests the feasibility to engineer autologous adenosine-releasing stem cells derived from a patient. Human embryonic stem cells (hESCs) have a high proliferative capacity and can be subjected to specific cellular differentiation pathways. hESCs, differentiated in vitro into neuroepithelial cells and grafted into the mouse brain, displayed intrahippocampal location and neuronal morphology. Using the same lentiviral micro-RNA vector, we demonstrated knockdown of ADK in hESCs. New developments and therapeutic challenges in using human mesenchymal stem cells and hESCs for epilepsy therapy will be critically evaluated.     

挫伤脑组织对人胚神经干细胞影响的体外研究

目的 建立人胚胎来源的神经干细胞体外培养方法，初步研究挫伤脑组织提取液(TBITE)对人胚神经干细胞(HNSCs)存活、增殖能力和分化的影响。方法 10周龄左右的胎儿大脑皮层细胞体外培养，神经干细胞增殖2～3代后，剔除生长因子，分别加入不同浓度的脑外伤病人的挫伤脑组织提取液，培养2～3周后，免疫细胞化学方法鉴定细胞分化后的类型。用Laemrnli凝胶电泳法分析培养液上清。结果成功获得以神经球形式生长的人胚神经干细胞，Nestin表达阳性；与空白对照相比，TBITE组中的神经干细胞有明显的增殖，并分化为神经细胞、星形胶质细胞和少突胶质细胞。随着TBITE含量的增加，星形胶质细胞的比例增加。结论 脑外伤的局部微环境可促进神经干细胞增殖和分化。     

Harnessing the stem cell properties of pericytes to repair the brain

Over the last ten years or so,it has become apparent that pericytes have the potential to differentiate into other cell types which may help in the repair and regeneration of tissue after injury.In fact,pericytes have been described as a precursor to mesenchymal stem cells.Their location at the interface between the microvasculature and the brain parenchyma means they are ideally positioned to initiate repair and regeneration in response to various factors.In this perspective,we will highlight how pericytes have stem cell potential alongside their role in regulating processes,such as angiogenesis and inflammation,and discuss how pericytes could be harnessed to promote tissue repair in the brain(Figure 1).     

Progress in clinical trials of stem cell therapy for cerebral palsy

Cerebral palsy is the most common disease in children associated with lifelong disability in many countries.Clinical research has demonstrated that traditional physiotherapy and rehabilitation therapies cannot alone cure cerebral palsy.Stem cell transplantation is an emerging therapy that has been applied in clinical trials for a variety of neurological diseases because of the regenerative and unlimited proliferative capacity of stem cells.In this review, we summarize the design schemes and results of these clinical trials.Our findings reveal great differences in population characteristics, stem cell types and doses, administration methods, and evaluation methods among the included clinical trials.Furthermore, we also assess the safety and efficacy of these clinical trials.We anticipate that our findings will advance the rational development of clinical trials of stem cell therapy for cerebral palsy and contribute to the clinical application of stem cells.     

染色体16三体人胚胎干细胞系的建立

背景：研究显示部分人胚胎干细胞系在建系或体外培养过程中出现染色体异常的现象,建立染色体异常的人胚胎干细胞系,可分析染色体对人胚胎干细胞特性的影响。 目的：拟建立染色体异常的人胚胎干细胞系。 设计、时间及地点：细胞学体内外观察,于2006-10/2008-02在南京大学医学院附属鼓楼医院生殖医学中心完成。 材料：冷冻人胚胎由南京大学医学院附属鼓楼医院生殖医学中心提供,4周龄NOD-scid鼠1只及ICR胎鼠成纤维细胞由南京大学医学院附属鼓楼医院实验动物中心提供。 方法：将人冷冻胚胎解冻后,置于囊胚培养基中微滴培养,胚胎发育至扩张囊胚期时分离囊胚中的内细胞团,以ICR胎鼠成纤维细胞作为饲养层,机械法传代,建立的人胚胎干细胞系命名为NJGLChES2。 主要观察指标：对10代以后的细胞进行核型分析,选择核型异常的人胚胎干细胞系鉴定其碱性磷酸酶和特异性标记物的表达,通过体外类胚体形成实验及体内畸胎瘤成瘤实验观察其分化能力。 结果：NJGLChES2人胚胎干细胞系核型分析结果呈16号染色体三体（47,XX,+16）,呈人胚胎干细胞克隆样生长,碱性磷酸酶和胚胎特异性标记物OCT-4,SSEA-3,SSEA-4,TRA-1-60,TRA-1-81的表达均为阳性。体外悬浮培养的NJGLChES2人胚胎干细胞可形成囊状拟胚体,体内接种至NOD-scid鼠腹股沟皮下后在接种部位可形成畸胎瘤,肿瘤组织含有来源于鳞状上皮、横纹肌和呼吸道黏膜上皮3个胚层的多种细胞类型。 结论：成功建立16号染色体三体人胚胎干细胞系NJGLChES2,可长期稳定增殖并保持未分化状态,且在体内外具有多向分化潜能。     

Human embryonic stem cell therapies for neurodegenerative diseases

There is a renewed enthusiasm for the clinical translation of human embryonic stem (hES) cells. This is abetted by putative clinically-compliant strategies for hES cell maintenance and directed differentiation, greater understanding of and accessibility to cells through formal cell registries and centralized cell banking for distribution, the revised US government policy on funding hES cell research, and paradoxically the discovery of induced pluripotent stem (iPS) cells. Additionally, as we consider the constraints (practical and fiscal) of delivering cell therapies for global healthcare, the more efficient and economical application of allogeneic vs autologous treatments will bolster the clinical entry of hES cell derivatives. Neurodegenerative disorders such as Parkinson's disease are primary candidates for hES cell therapy, although there are significant hurdles to be overcome. The present review considers key advances and challenges to translating hES cells into novel therapies for neurodegenerative diseases, with special consideration given to Parkinson's disease and Alzheimer's disease. Importantly, despite the focus on degenerative brain disorders and hES cells, many of the issues canvassed by this review are relevant to systemic application of hES cells and other pluripotent stem cells such as iPS cells.     

神经干细胞移植在脑损伤治疗中的应用

背景：成年鼠脑纹状体中已分离出具有分化成神经元及各种胶质细胞潜能的神经干细胞后,医学工作者试图从中寻找新思路用于脑损伤的治疗。目的：总结神经干细胞移植对治疗脑损伤中的作用及其前景。方法：电子检索EMbase(1980-01/2011-04),MEDLINE(1966-01/2011-04),中国生物医学文献数据库(CBM,1978/2011-04)和中国期刊全文数据库(CNKI),筛查相关文章的参考文献。中文检索词“神经干细胞,脑损伤,脑卒中,脑梗死”,英文检索词“neural stem cells, brain injury, stroke, cerebral infarction”,最终纳入符合标准的文献12篇。结果与结论：动物研究结果显示神经干细胞移植可以改善神经功能并且明显降低脑组织炎症反应,沉默NgR基因、GDNF基因修饰和联合移植BDNF可更大程度上恢复神经功能。临床试验发现神经干细胞移植能在一定程度上改善脑卒中患者的后遗症。提示神经干细胞移植在动物实验中获取明显效果,可进一步应用于临床并研究其疗效。     

人胚胎干细胞的保存方法*☆

目的：胚胎干细胞经传代长期培养后会导致核型出现异常、未分化细胞的克隆数减少，因此经低温保存后复苏的胚胎干细胞必须要保持其未分化状态及生物学特性。实验拟比较程序降温法、改良慢速冻存法及传统慢速冻存法对人胚胎干细胞冷冻保存的效率，以及解冻后细胞生长与分化的状态。 方法：实验于2006-07/2007-04在解放军军事医学科学院野战输血研究所完成。①材料：清洁级孕13.5 d的ICR小鼠由军事医学科学院动物中心提供，用于制备鼠成纤维细胞饲养层，实验过程中对动物的处置符合动物伦理学标准。人胚胎干细胞系PKU由北京大学第三医院生殖中心建立并提供；kryo-550-16程序降温仪为英国planner公司产品，由液氮压力泵、液氮罐、层流冷冻箱、温度传感器组成。 ②实验方法：将人胚胎干细胞系PKU接种在胚胎鼠成纤维细胞饲养层上，胶原酶Ⅳ消化传代，分3组冷冻保存人胚胎干细胞。程序降温法：用吸管将消化后人胚胎干细胞吹成若干团块，取65个团块移入配制的冻存液中，装入麦管进行三段式降温，即22 ℃~-7 ℃，每分钟降温2.5 ℃→-7 ℃置核后停留5 min→-7 ℃~-30 ℃，每分钟降温0.3 ℃→-30 ℃~-150 ℃，每分钟降温10 ℃。降至-150 ℃时置液氮中保存。改良慢速冻存法：A管含人胚胎干细胞培养基和血清替代品，B管含人胚胎干细胞培养基和二甲亚砜，将60个团块置入A管，再将B管液体移入A管，混匀后-70 ℃过夜，第2天移入液氮中保存。传统慢速冻存法：将60个团块直接放入添加二甲亚砜的人胚胎干细胞培养基中混匀，分装入冻存管，-4 ℃放置1 h，-70 ℃过夜，第2天移入液氮长期保存。③实验评估：解冻后，将保持完整形态且细胞未散离的人胚胎干细胞团块回收，比较3组细胞复苏率、复制生长状态、克隆分化程度。鉴定程序降温组人胚胎干细胞的生物学特性。 结果：①复苏率：程序降温组、改良慢速冻存组、传统慢速冻存组的细胞复苏率分别为78.5%、56.7%和23.3%，组间比较差异有显著性意义（x2=6.81~13.89，P < 0.01）。②复制生长与克隆分化程度：解冻后第7天与传统慢速冻存组比较，程序降温组及改良慢速冻存组细胞克隆均明显增大（t=7.36，P < 0.05；t=6.89，P < 0.05），且解冻后克隆不易分化（x2=20.47，P < 0.01；x2=9.69，P < 0.01）。③生物学特性鉴定：程序降温组解冻后第10代人胚胎干细胞染色体核型正常，人胚胎干细胞特异性表面抗原SSEA-4及TRI-1-81表达呈阳性，RT-PCR可检测到nanog基因表达，SSEA-4及OCT-4阳性表达率分别为83.44%、89.38%。 结论：①程序降温法是冷冻保存人胚胎干细胞的有效方法，解冻后的干细胞在继续培养过程中仍可保持其正常核型和生物学特性。②在不具备程序降温仪的条件下，采用改良慢速冻存法保存胚胎干细胞也是可行的。     

Differentiation of human dopamine neurons from an embryonic carcinomal stem cell line

Previous studies from this laboratory have demonstrated that fibroblast growth factor 1 together with a number of co-activator molecules (dopamine, TPA, IBMX/forskolin), will induce the expression of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) in 10% of human neurons (hNTs) derived from the NT2 cell line [10]. In the present study, we found that TH induction was increased to nearly 75% in hNTs when cells were permitted to age 2 weeks in culture prior to treatment with the differentiation cocktail. This high level of TH expression was sustained 7 days after removal of the differentiating agents from the media. Moreover, the induced TH present in these cells was enzymatically active, resulting in the production of low levels of dopamine (DA) and its metabolite DOPAC. These findings suggest that hNTs may provide an important tissue culture model for the study of factors regulating TH gene expression in human neurons. Moreover, hNTs may serve, in vivo, as a source of human DA neurons for use in transplantation therapies.     

Neuronal circuitry reconstruction after stem cell therapy in damaged brain

Transplantation of neuronal precursors derived from human pluripotent stem cells is a promising therapy for the treatment of neurological disorders associated with neuronal loss,such us neurodegenerative diseases,brain trauma and stroke.The functional integration of grafted neurons differentiated from stem cells into the host injured neuronal circuitry has been a major challenge in cell therapy strategies for brain repair(Palma-Tortosa et al.,2021).Even though other cell types or mechanisms may provide modest clinical improvements,neuronal replacement and reconstruction of the damaged area are crucial for an optimal and long-term recovery.     

The potential of stem cell-based therapy for retinal repair

<正>Like injured neurons in the brain or spinal cord,neurons in the retina are incapable to regenerate following injury and ultimately would lead to irreversible neuronal loss and vision impairment.Over decades,extensive effort has been made to develop strategies to protect retinal neurons from death;however,the outcome is     

干细胞移植治疗缺血性卒中的研究进展及临床相关问题

脑血管病与心脏病、恶性肿瘤构成人类的三大致死性疾病。其中,脑卒中是指由于急性脑循环障碍所致的局限性或全面性神经功能缺损综合征,也称急性脑血管事件。脑卒中发病率、病死率和病残率逐年升高,生存者中50%～70%遗留严重残疾,给社会和家庭带来沉重负担。现有的治疗措施尚未显示出明显的临床治疗效果,而干细胞移植治疗在动物实验和临床研究中取得的初步成果及其对     

小鼠胚胎干细胞体外向神经干细胞的分化研究

目的观察无血清培养法诱导小鼠胚胎干细胞(Embryome stem cell,ESCs)ESCs体外分化为神经干细胞(Neural stem cell,NSCs)的诱导效率.方法采用无血清培养法诱导小鼠ESCs体外分化为NSCs,对诱导不同时间点的细胞行免疫荧光,RT-PCR检测nestin的表达.结果诱导48 h开始出现nestinmRNA的表达,诱导72 h出现nestin蛋白表达.诱导120 h nestin的表达达高峰,阳性细胞率为(70.3±8.02)%.Nestin阳性细胞呈球形生长,可不断增殖.结论本诱导方法可获得较高纯度的NSCs,所获得的神经干细胞与脑内来源的NSCs有相似的生物学特性.