Human neural stem cells genetically modified for brain repair in neurological disorders

Existence of multipotent neural stem cells (NSC) has been known in developing or adult mammalian CNS, including humans. NSC have the capacity to grow indefinitely and have multipotent potential to differentiate into three major cell types of CNS, neurons, astrocytes and oligodendrocytes. Stable clonal lines of human NSC have recently been generated from the human fetal telencephalon using a retroviral vector encoding v‐myc. One of the NSC lines, HB1.F3, carries normal human karyotype of 46XX and has the ability to self‐renew, differentiate into cells of neuronal and glial lineages, and integrate into the damaged CNS loci upon transplantation into the brain of animal models of Parkinson disease, HD, stroke and mucopolysaccharidosis. F3 human NSC were genetically engineered to produce L‐dihydroxyphenylalanine (L‐DOPA) by double transfection with cDNA for tyrosine hydroxylase and guanosine triphosphate cylohydrolase‐1, and transplantation of these cells in the brain of Parkinson disease model rats led to L‐DOPA production and functional recovery. Proactively transplanted F3 human NSC in rat striatum, supported the survival of host striatal neurons against neuronal injury caused by 3‐nitropro‐pionic acid in rat model of HD. Intravenously introduced through the tail vein, F3 human NSC were found to migrate into ischemic lesion sites, differentiate into neurons and glial cells, and improve functional deficits in rat stroke models. These results indicate that human NSC should be an ideal vehicle for cell replacement and gene transfer therapy for patients with neurological diseases. In addition to immortalized human NSC, immortalized human bone marrow mesenchymal stem cell lines have been generated from human embryonic bone marrow tissues with retroviral vectors encording v‐myc or teromerase gene. These immortalized cell lines of human bone marrow mesenchymal stem cells differentiated into neurons/glial cells, bone, cartilage and adipose tissue when they were grown in selective inducing media. There is further need for investigation into the neurogenic potential of the human bone marrow stem cell lines and their utility in animal models of neurological diseases.     

Brain transplantation of genetically engineered human neural stem cells globally corrects brain lesions in the mucopolysaccharidosis type VII mouse

In the present study, we investigated the feasibility of using human neural stem cells (NSCs) in the treatment of diffuse central nervous system (CNS) alterations in a murine model of mucopolysaccharidosis VII (MPS VII), a lysosomal storage disease caused by a genetic defect in the beta-glucuronidase gene. An immortalized NSC line derived from human fetal telencephalon was genetically engineered to overexpress beta-glucuronidase and transplanted into the cerebral ventricles of neonatal MPS VII mouse. Transplanted human NSCs were found to integrate and migrate in the host brain and to produce large amount of beta-glucuronidase. Brain contents of the substrates of beta-glucuronidase were reduced to nearly normal levels, and widespread clearing of lysosomal storage was observed in the MPS VII mouse brain at 25 days posttransplantation. The number of engrafted cells decreased markedly after the transplantation, and it appears that the major cause of the cell death was not the immune response of the host but apoptotic cell death of grafted human NSCs. Results showed that human NSCs would serve as a useful gene transfer vehicle for the treatment of diffuse CNS lesions in human lysosomal storage diseases and are potentially applicable in the treatment of patients suffering from neurological disorders.     

人胚神经干细胞体外长期培养系统的建立

目的建立人胚神经干细胞的体外长期培养系统并研究其生物学特性.方法从胚龄为20周的人胚胎皮质组织分离出神经干细胞,连续传代培养并检测生长曲线,测定长期培养对细胞周期和细胞分化及冻存的影响.结果体外培养神经干细胞达到了10个月,传代25代,总的细胞数增加了105倍.干细胞只有培养1个月后才能有效去除前体细胞.细胞周期显示细胞保持了活跃的增殖,多次传代后细胞冻存对细胞的存活力无明显影响,多向分化潜力表现出稳定性.结论人神经干细胞可于体外长期培养,这也是细胞纯化的有效方法.     

Brain transplantation of human neural stem cells transduced with tyrosine hydroxylase and GTP cyclohydrolase 1 provides functional improvement in animal models of Parkinson disease

Parkinson disease is a neurodegenerative disease characterized by loss of midbrain dopaminergic neurons resulting in movement disorder. Neural stem cells (NSC) of the CNS have recently aroused a great deal of interest, not only because of their importance in basic research of neural development, but also for their therapeutic potential in neurological disorders. We have recently generated an immortalized human NSC cell line, HB1.F3, via retrovirus‐mediated v‐myc transfer. This line is capable of self‐renewal, is multipotent, and expresses cell specific markers for NSC, ATP‐binding cassettes transporter (ABCG2) and nestin. Next, we co‐transduced the F3 NSC line with genes encoding tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GTPCH1) in order to generate dopamine‐producing NSC. The F3.TH.GTPCH human NSC line expresses TH and GTPCH phenotypes as determined by RT‐PCR, western blotting and immunocytochemistry, and shows a 800 to 2000‐fold increase in production of l ‐dihydroxyphenyl alanine in HPLC analysis. A marked improvement in amphetamine‐induced turning behavior was observed in parkinsonian rats implanted with F3.TH.GTPCH cells, but not in control rats receiving F3 NSC. In the animals showing functional improvement, a large number of TH‐positive F3.TH.GTPCH NSC were found at injection sites. These results indicate that human NSC, genetically transduced with TH and GTPCH1 genes, have great potential in clinical utility for cell replacement therapy in patients suffering from Parkinson disease.     

立体定向神经干细胞移植治疗脑出血后遗症疗效分析

目的观察应用神经干细胞移植治疗脑出血后遗症的临床疗效。方法2000年6月至2006年12月我们对16例脑出血患者采用立体定向技术，选取病变侧基底节区作为靶点进行神经干细胞移植术，于治疗前、治疗后1月、6月进行神经功能评分，应用国际通用的功能独立性测量评定其运动及生活能力。结果接受神经干细胞移植的脑出血患者无严重手术并发症。手术后6个月内临床症状改善有效率达81．25％（13例／16例），本组16例术前FIM评分为90．21±2．32，术后1个月为92．76±1．89，术后6个月为96．37±3．83，差异有显著性意义。治疗1个月后功能独立性评分高于治疗前（P〈0．05），而治疗6个月后高于1个月（P〈0．05）。结论神经千细胞移植治疗可以一定程度地改善脑出血患者的后遗症，可提高脑出血患者的运动功能，提高患者生活质量，但其长期疗效仍需进一步观察。     

人胚脑与脊髓神经干细胞体外生物学特性的差异

目的:探讨人胚脑源性神经干细胞和脊髓源性神经干细胞的体外培养和分化的差异。方法:从人胚脑组织和脊髓组织中分离培养神经干细胞,分为EGF组、bFGF组、EGF±bFGF组,在连续传代过程中观察并比较神经干细胞体外培养特性的差异:用血清诱导神经干细胞分化,观察其分化状况的不同。结果:从人胚脑组织分离的细胞在bFGF 单独存在时无法形成神经球,在EGF或EGF±bFGF存在时形成大量具有连续增殖能力的神经球;从人胚脊髓组织分离的细胞在EGF单独存在时无法形成神经球,在bFGF单独存在时只形成少量神经球,在EGF±bFGF存在时形成大量具有连续增殖能力的神经球。同样在EGF±bFGF存在的情况下,脑源性于细胞的增殖速度较快。经血清诱导后,脑组织来源的干细胞分化为NSE阳性细胞数明显多于脊髓组织来源的干细胞,二者之间的差异具有显著性(P<0．05)。结论:脑源性和脊髓源性神经干细胞在生长和分化方面有明显差别:脑源性神经干细胞可在bFGF或EGF士bFGF存在的情况下长期传代,而脊髓源性神经干细胞只能在EGF±bFGF存在的情况下长期传代,脑源性干细胞的增殖能力明显高于脊髓源性干细胞;脑源性干细胞较脊髓源性干细胞更易分化为神经元。     

Similarity on neural stem cells and brain tumor stem cells in transgenic brain tumor mouse models

Although it is believed that glioma is derived from brain tumor stem cells, the source and molecular signal pathways of these cells are still unclear. In this study, we used stable doxycycline-inducible transgenic mouse brain tumor models (c-myc⁺/SV40Tag⁺/Tet-on⁺) to explore the malignant trans-formation potential of neural stem cells by observing the differences of neural stem cells and brain tumor stem cells in the tumor models. Results showed that chromosome instability occurred in brain tumor stem cells. The numbers of cytolysosomes and autophagosomes in brain tumor stem cells and induced neural stem cells were lower and the proliferative activity was obviously stronger than that in normal neural stem cells. Normal neural stem cells could differentiate into glial fibrillary acidic protein-positive and microtubule associated protein-2-positive cells, which were also negative for nestin. However, glial fibrillary acidic protein/nestin, microtubule associated protein-2/nestin, and glial fibrillary acidic protein/microtubule associated protein-2 double-positive cells were found in induced neural stem cells and brain tumor stem cells. Results indicate that induced neural stem cells are similar to brain tumor stem cells, and are possibly the source of brain tumor stem cells.     

脑肿瘤干细胞与胶质瘤生物治疗研究进展

脑肿瘤是中枢神经系统常见疾病之一,对人类健康乃至生命的危害极大。脑肿瘤发病率约占全身肿瘤的5％,占儿童肿瘤的70％,且近年来仍呈上升趋势。其中以胶质细胞瘤发病率最高,约占脑肿瘤总数的40.49％,综合发病年龄高峰位于30～40岁,或10～20岁。     

室管膜下区与脑肿瘤干细胞的研究现状

脑肿瘤细胞的起源一直是学者们关注的焦点问题之一。研究发现,脑肿瘤中存在少量具有干细胞特性的细胞即脑肿瘤干细胞(brain tumor stem cells,BTSCs),它们是引发肿瘤并维持其生长的细胞来源[l]。BTSCs与神经干细胞(neural stem cells,NSCs)存在诸多相似性。成人侧脑室室管膜下区(subventricular zone,SVZ)是NSCs最集中的部位。目前,寻找胶质瘤和SVZ区的相关性以便解释脑胶质瘤的起源之谜是国内外研究的热点问题,本文将对其作以如下综述。     

Induced neural stem/precursor cells for fundamental studies and potential application in neurodegenerative diseases

Recent research has shown that defined sets of exogenous factors are sufficient to convert rodent and human somatic cells directly into induced neural stem cells or neural precursor cells(iNSCs/iNPCs).The process of transdifferentiation bypasses the step of a pluripotent state and reduces the risk of tumorigenesis and genetic instability while retaining the self-renewing capacity.This iNSC/iNPC technology has fueled much excitement in regenerative medicine,as these cells can be differentiated into target cells for replacement therapy for neurodegenerative diseases.Patients' somatic cell-derived iNSCs/iNPCs have also been proposed to serve as disease models with potential value in both fundamental studies and clinical applications.This review focuses on the mechanisms,techniques,and applications of iNSCs/iNPCs from a series of related studies,as well as further efforts in designing novel strategies using iNSC/iNPC technology and its potential applications in neurodegenerative diseases.     

Spatio-temporal dynamics, differentiation and viability of human neural stem cells after implantation into neonatal rat brain

Neural stem cells (NSCs) have attracted major research interest due to their potential use in cell replacement therapy. In patients, human cells are the preferred choice, one source of human NSCs being the brain of fetuses. The aims of the present study were to explore the long‐term differentiation, mobility and viability of NSCs derived from the human fetal striatum in response to intracerebral implantation. To investigate long‐term spatio‐temporal and functional dynamics of grafts in vivo by magnetic resonance imaging, these cells were labeled with superparamagnetic iron oxide (SPIO) nanoparticles prior to implantation. SPIO‐labeling of human NSCs left the quantitative profile of the proliferation, cell composition and differentiation capacity of the cells in vitro unaltered. Also after transplantation, the phenotypes after long‐term cell differentiation were not significantly different from naïve cells. Upon transplantation, we detected a hypointensity corresponding to the striatal graft location in all animals and persisting for at least 4 months. The hypointense signal appeared visually similar both in location and in volume over time. However, quantitative volumetric analysis showed that the detectable, apparent graft volume decreased significantly from 3 to 16 weeks. Finally, the human NSCs were not proliferating after implantation, indicating lack of tumor formation. These cells are thus a promising candidate for translationally relevant investigations for stem cell‐based regenerative therapies.     

Stem cell transplantation for treatment of cerebral ischemia in rats Effects of human umbilical cord blood stem cells and human neural stem cells

BACKGROUND:Exogenous neural stem cell transplantation promotes neural regeneration. However, various types of stem cells transplantation outcomes remain controversial. OBJECTIVE:To explore distribution, proliferation and differentiation of human neural stem cells (hNSCs) and human umbilical cord blood stem cells (hUCBSCs) following transplantation in ischemic brain tissue of rats, and to compare therapeutic outcomes between hNSCs and hUCBSCs. DESIGN, TIME AND SETTING:Randomized controlled animal studies were performed at the Experimental Animal Center of Nanjing Medical University and Central Laboratory of Second Affiliated Hospital of Nanjing Medical University of China from September 2008 to April 2009. MATERIALS:hNSCs were harvested from brain tissue of 10-13 week old fetuses following spontaneous abortion, and hUCBSCs were collected from umbilical cord blood of full-term newborns at the Second Affiliated Hospital of Nanjing Medical University of China. hNSCs and hUCBSCs were labeled by 5-bromodeoxyuridine (BrdU) prior to transplantation. METHODS:Rat models of cerebral ischemia were established by the suture method. A total of 60 healthy male Sprague Dawley rats aged 7-9 weeks were randomly assigned to hNSC transplantation, hUCBSC transplantation and control groups. The rat models in the hNSC transplantation, hUCBSC transplantation and control groups were infused with hNSC suspension, hUCBSC suspension and saline via the caudal vein, respectively. MAIN OUTCOME MEASURES:The distribution, proliferation and differentiation of hNSCs and hUCBSCs in ischemic brain tissue were observed using immunohistochemical methods. Neurological function in rats was assessed using the neurological severity score. RESULTS:The number of BrdU-positive cells was significantly greater in the hNSC transplantation group compared with hUCBSC transplantation group at 14 days following transplantation (P < 0.05). The number of BrdU-positive cells reached a peak at 28 days following transplantation. Nestin-positive, glial fibrillary acidic protein-positive, cyclic nucleotide 3' phosphohydrolase-positive and neuron specific enolase-positive cells were visible following transplantation. No significant difference was determined in the constituent ratio of various cells between hNSC and hUCBSC transplantation groups (P > 0.05). The neurological severity score was significantly decreased in rats at 21 days following transplantation (P < 0.05). No significant difference was detected in neurological severity score between hNSC and hUCBSC transplantation groups at various time points (P > 0.05). CONCLUSION:The transplanted hNSCs and hUCBSCs can migrate into ischemic brain tissue, proliferate and differentiate into neuron-like, astrocyte-like and oligodendrocyte-like cells, and improve neurological function in rats with cerebral ischemia.     

Estrogen promotes differentiation and survival of dopaminergic neurons derived from human neural stem cells

To investigate the effect of estrogen on neuronal differentiation, especially on dopaminergic (DA) neurons, human neural stem cells (NSCs) were differentiated in the presence of 17beta-estradiol. NSCs gave rise to tyrosine hydroxylase (TH)-positive neurons in vitro, the proportion of which was increased by 17beta-estradiol. Increase in TH-positive neurons was abrogated by an estrogen receptor (ER) antagonist, ICI182780, suggesting ERs play a role in differentiation of DA neurons. The observation that ERs were expressed in both proliferating NSCs and postmitotic DA neurons suggested that increase in TH-positive neurons was due to induction and support of DA neurons. 17beta-Estradiol also increased the number of DA neurons derived from human NSCs in vivo when the cells were grafted into mouse brains. These results support a possible role for estrogen in the transplantation of NSCs for Parkinson's disease.     

Effect of neurosphere size on the growth rate of human neural stem/progenitor cells

Neural stem/progenitor cells (NSPCs) proliferate as aggregates in vitro, but the mechanism of aggregation is not fully understood. Here, we report that aggregation promotes the proliferation of NSPCs. We found that the proliferation rate was linear and depended on the size of the aggregate; that is, the population doubling time of the NSPCs gradually decreased as the diameter approached 250 micro m and flattened to a nearly constant value beyond this diameter. Given this finding, and with the intent of enhancing the efficiency of human NSPC expansion, we induced the NSPCs to form aggregates close to 250 micro m in diameter quickly by culturing them in plates with U-bottomed wells. The NSPCs formed aggregates effectively in the U-bottomed wells, with cell numbers approximately 1.5 times greater than those in the aggregates that formed spontaneously in flat-bottomed wells. In addition, this effect of aggregation involved cell-cell signaling molecules of the Notch1 pathway. In the U-bottomed wells, Hes1 and Hes5, which are target genes of the Notch signal, were expressed at higher levels than in the control, flat-bottomed wells. The amount of cleaved Notch1 was also higher in the cells cultured in the U-bottomed wells. The addition of gamma-secretase inhibitor, which blocks Notch signaling, suppressed cell proliferation in the U-bottomed wells. These results suggest that the three-dimensional architecture of NSPC aggregates would create a microenvironment that promotes the proliferation of human NSPCs.     

Soluble mediators from human neural stem cells play a critical role in suppression of T‐cell activation and proliferation

Human neural stem cells (hNSCs) can control inflammation in the central nervous system, although the underlying mechanisms are not understood fully. We investigated the immunomodulatory effect of hNSCs on human T cells and the underlying mechanisms. Culture supernatant from an immortalized hNSC cell line, HB1.F3, which has a therapeutic effect on acute stroke and intracerebral hemorrhage, suppressed the proliferation of allogeneically or mitogenically stimulated human peripheral T cells, including the CD3⁺CD103⁺ subpopulation. CFSE labeling and flow cytometry showed that the suppression of proliferation was caused by cell cycle arrest and induction of apoptosis. The lack of significant change in caspase‐8 levels and the significant reduction in Bcl‐2 expression in the affected T cells suggest that the intrinsic pathway plays a major role in soluble‐factor‐mediated T‐cell apoptosis. The addition of culture supernatant from hNSCs to activated T cells reduced the expression of the activation markers CD69 and CD25 at 24 hr after activation, but at 48 hr only CD69 was down‐regulated. A cytometry bead assay showed that the secretion of interleukin (IL)‐2 decreased significantly, whereas that of IL‐4, IL‐10, tumor necrosis factor‐α, and interferon‐γ increased. These results show that hNSCs can negatively affect human peripheral T cells by suppressing their activation and proliferation through soluble mediators, suggesting that hNSCs have a bystander immunomodulatory effect on T cells. © 2009 Wiley‐Liss, Inc.     

经枕大池神经干细胞移植治疗大鼠创伤性脑损伤的研究

目的 将神经干细胞经枕大池移植到创伤性脑损伤模型大鼠蛛网膜下腔中并观察其存活、迁移和分化,从而为神经干细胞的体内存活、迁移和分化机理研究和临床应用提供实验依据.方法 体外培养BrdU标记的胚胎神经干细胞并应用免疫荧光细胞化学染色对BrdU、神经干细胞标记物nestin的表达进行鉴定:采用Feeney自由落体撞击法制做大鼠脑损伤模型,伤后24 h将BrdU标记的胚胎神经十细胞经立体定向注射移植到蛛网膜下腔;制作大鼠脑绢织石蜡切片,应用免疫组织化学染色检测BrdU、微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)表达;伤前24h、伤后24 h及1、2周行动物运动神经功能评分.结果 免疫荧光检测显示神经球的表面细胞表达nestin及BrdU:免疫组织化学染色检测到脑内损伤灶存在BrdU阳性神经干细胞、MAP2阳性神经元和GFAP阳性胶质细胞;接受神经十细胞移植的大鼠神经运动功能评分的恢复较对照组有明显提高,差异有统计学意义(P<0.05).结论 经枕大池移植到脑损伤大鼠蛛网膜下腔中的神经干细胞能存活且具有远距离迁移能力,并明显有助于脑损伤大鼠神经运动功能的恢复.     

人胚神经干细胞移植治疗大鼠脑缺血的实验研究

目的　研究人胚神经干细胞(hNSCs)移植治疗脑缺血大鼠的效果及其在缺血大鼠脑内的状况。方法　从自然流产的孕10~13周的人胚脑组织中分离、培养神经干细胞。采用线栓法制作大鼠脑缺血模型,1d后经尾静脉移植未分化的hNSCs入脑缺血大鼠体内,对移植后大鼠进行神经损害严重程度评分(NSS),用免疫组化方法观察移植后hNSCs的存活、迁徙、分化状况。结果　从人胎脑中成功培养出hNSCs,培养条件下呈悬浮状态生长,形成神经球,绝大多数的细胞表达神经干细胞的标记物神经巢蛋白(nestin)。hNSCs移植组大鼠自移植后3周末起其NSS显著低于对照组(P<0 .05);移植后2、3、4、5周脑组织切片中均可见5 溴脱氧嘧啶尿苷(Brdu)染色阳性细胞,缺血侧明显多于对侧(P<0 .05),移植后3、4、5周末明显多于移植后2周(均P<0 .05);移植组各时间点脑组织切片中均可见nestin染色阳性细胞;在Brdu阳性细胞群中, 73 8%为胶质纤维酸性蛋白(GFAP)染色阳性的星形胶质细胞, 16 7%为2, 3 环核苷酸磷酸二脂酶(CNPase)染色阳性的少突胶质细胞, 9 5%为神经元特异性烯醇化酶(NSE)染色阳性的神经元。结论　经静脉移植hNSCs能有效改善脑梗死动物的神经功能,hNSCs体内体外均具有多向分化潜能,受缺血部位微环境信号的影响分化成3种主要类型的神经细胞。     

小鼠胚胎干细胞诱导的神经前体细胞纹状体移植对PD大鼠的治疗作用

目的 观察来源于小鼠胚胎干细胞的神经前体细胞移植PD大鼠纹状体后的存活、分化以及细胞移植对PD大鼠的治疗作用。方法 采用无血清方法将小鼠胚胎干细胞定向诱导为神经前体细胞，免疫组化技术观察移植细胞的存活、分化。结果 胚胎体在N2选择性培养基选择生长5d后，85％以上的小鼠胚胎干细胞分化为nestin阳性的神经前体细胞。移植到PD大鼠纹状体后大部分神经前体细胞存活良好，移植细胞分别保持为未分化的nestin阳性的神经前体细胞和TH阳性的神经元。移植后3周，PD大鼠的旋转次数明显减少。结论 胚胎干细胞来源的神经前体细胞移植PD大鼠纹状体后能分化为TH阳性的神经细胞，对PD有治疗作用。