Clinical Trials of Adult Stem Cell Therapy in Patients with Ischemic Stroke

Stem cell therapy is considered a potential regenerative strategy for patients with neurologic deficits. Studies involving animal models of ischemic stroke have shown that stem cells transplanted into the brain can lead to functional improvement. With current advances in the understanding regarding the effects of introducing stem cells and their mechanisms of action, several clinical trials of stem cell therapy have been conducted in patients with stroke since 2005, including studies using mesenchymal stem cells, bone marrow mononuclear cells, and neural stem/progenitor cells. In addition, several clinical trials of the use of adult stem cells to treat ischemic stroke are ongoing. This review presents the status of our understanding of adult stem cells and results from clinical trials, and introduces ongoing clinical studies of adult stem cell therapy in the field of stroke.     

Therapeutic time window and dose response of autologous bone marrow mononuclear cells for ischemic stroke

Although mononuclear cells (MNCs) from bone marrow are being investigated in phase I clinical trials in stroke patients, dose response, therapeutic time window, and biodistribiton have not been well‐characterized in animal stroke models. Long Evans rats underwent common carotid artery/middle cerebral artery occlusion (CCA/MCAo) and 24 hr later were randomized to receive saline IV or a bone marrow aspiration followed by an IV infusion of autologous separated MNCs (1 million, 10 million, or 30 million cells/kg). In another experiment, rats underwent CCAo/MCAo and were randomized at 24 hr, 72 hr, or 7 days after stroke to receive a saline injection or 10 million/kg MNCs. All animals were evaluated on the cylinder and corner tests up to 28 days. MNCs were tracked using Q‐dot nanocrystals to monitor biodistribution. Animals treated with MNCs at 10 million and 30 million cells/kg at 24 hr after stroke had significant reductions in neurological deficits and lesion size compared with saline controls or animals treated with 1 million cells/kg. There was no difference in neurological deficits in the 10 and 30 million cell/kg groups at 28 days. Animals treated with MNCs at 72 hr but not at 7 days showed a significant reduction in neurological deficits by 28 days. Labeled MNCs were found in the brain, spleen, lung, liver, and kidney at 1 hr and exponentially decreased over the ensuing week. In conclusion, we found a maximum reduction in neurological deficits at 10 and 30 million cells/kg and a therapeutic time window up to 72 hr after stroke. © 2011 Wiley‐Liss, Inc.     

自体外周造血干细胞移植治疗进展型多发性硬化的临床观察(附一例报告)

目的观察自体外周造血干细胞移植(auto-PBSCT)治疗进展型多发性硬化(PMS)的疗效及安全性。方法PMS 1例,以重组人粒细胞集落刺激因子(G-CSF)动员造血干细胞(HSC),血细胞分离仪分离外周血干细胞50 mL,按患者体重拟回输的CD34 细胞2.85×106个/kg于-80℃液氮冻存。在马利兰/环磷酰胺方案预处理后,经锁骨下静脉回输HSC,G-CSF协助造血恢复。移植后2、4、8、12周观察患者临床、体征、实验室及影像学指标。结果移植后患者临床症状及体征基本恢复,MRI显示病灶有所缩小。结论auto-PBSCT治疗PMS较为安全、有效。     

Endovascular Therapy for Ischemic Stroke

The utility of intravenous tissue plasminogen activator (IV t-PA) in improving the clinical outcomes after acute ischemic stroke has been well demonstrated in past clinical trials. Though multiple initial small series of endovascular stroke therapy had shown good outcomes as compared to IV t-PA, a similar beneficial effect had not been translated in multiple randomized clinical trials of endovascular stroke therapy. Over the same time, there have been parallel advances in imaging technology and better understanding and utility of the imaging in therapy of acute stroke. In this review, we will discuss the evolution of endovascular stroke therapy followed by a discussion of the key factors that have to be considered during endovascular stroke therapy and directions for future endovascular stroke trials.     

序贯式干细胞移植术治疗假肥大型肌营养不良症临床研究

目的：观察假肥大型肌营养不良症（PMD）患儿接受序贯式干细胞移植术（SCT）后抗肌萎缩蛋白（dystrophin）的表达、dystrophin基因和运动功能的变化。方法：采用自身对照法,2008年2月至2010年11月对5例8～14岁男性PMD患儿接受序贯式SCT[即依次进行脐带间充质干细胞（UCMSC）经静脉内移植-UCMSC肌肉内移植-单倍体相合造血干细胞移植术（Haplo-HSCT）]治疗,观察患儿血清酶学、基因分析、肌电图、肌肉活检及肌萎缩蛋白、造血重建的植入证据的变化。结果：移植后①血清肌酸激酶数值显著降低;②PCR-STR检测4例为完全供者型嵌合,1例3/6位点相合患儿未植入为完全受者型;③4例外显子缺失患儿外周血及骨髓表达正常基因型;④肌肉活检显示供受者嵌合状态,缺失的外显子弱阳性表达。肌细胞形态改善,肌萎缩蛋白间断弱阳性表达;⑤肌力及运动功能较治疗前无减退或改善。结论：PMD患儿接受序贯式SCT后缺失的外显子转变为正常基因型,肌细胞膜有肌萎缩蛋白阳性或弱阳性表达,可提高患儿的运动功能。     

Recent Stem Cell Research on Hemorrhagic Stroke : An Update

Although technological advances and clinical studies on stem cells have been increasingly reported in stroke, research targeting hemorrhagic stroke is still lacking compared to that targeting ischemic stroke. Studies on hemorrhagic stroke are also being conducted, mainly in the USA and China. However, little research has been conducted in Korea. In reality, stem cell research or treatment is unfamiliar to many domestic neurosurgeons. Nevertheless, given the increased interest in regenerative medicine and the increase of life expectancy, attention should be paid to this topic. In this paper, we summarized pre-clinical rodent studies and clinical trials using stem cells for hemorrhagic stroke. In addition, we discussed results of domestic investigations and future perspectives on stem cell research for a better understanding.     

Cellular Treatments for Spinal Cord Injury: The Time is Right for Clinical Trials

More than 1 million people in the United States live with a spinal cord injury (SCI). Despite medical advances, many patients with SCIs still experience substantial neurological disability, with loss of motor, sensory, and autonomic function. Cell therapy is ideally suited to address the multifactorial nature of the secondary events following SCI. Remarkable advances in our understanding of the pathophysiology of SCI, structural and functional magnetic resonance imaging, image-guided micro-neurosurgical techniques, and transplantable cell biology have enabled the use of cell-based regenerative techniques in the clinic. It is important to note that there are more than a dozen recently completed, ongoing, or recruiting cell therapy clinical trials for SCI that reflect the views of many key stakeholders. The field of regenerative neuroscience has reached a stage in which the clinical trials are scientifically and ethically justified. Although experimental models and analysis methods and techniques continue to evolve, no model will completely replicate the human condition. It is recognized that more work with cervical models of contusive/compressive SCI are required in parallel with clinical trials. It is also important that the clinical translation of advances made through well-established and validated experimental approaches in animal models move forward to meet the compelling needs of individuals with SCI and to advance the field of regenerative neuroscience. However, it is imperative that such efforts at translation be done in the most rigorous and informed fashion to determine safety and possible efficacy, and to provide key information to clinicians and basic scientists, which will allow improvements in regenerative techniques and the validation and refinement of existing preclinical animal models and research approaches. The field of regenerative neuroscience should not be stalled at the animal model stage, but instead the clinical trials need to be focused, safe, and ethical, backed up by a robust, translationally relevant preclinical research strategy.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-011-0076-7) contains supplementary material, which is available to authorized users.     

Expression of CD34 and other haematopoietic antigens on neuroblastoma cells: consequences for autologous bone marrow and peripheral blood stem cell transplantation

Autologous peripheral blood stem cells, obtained by CD34⁺ stem cell selection, are being used with increasing frequency for transplantation in patients with neuroblastoma. Here, we examined the surface membrane antigens of neuroblastoma cells with a panel of hematopoietic monoclonal antibodies (mAbs), including anti-CD34 mAbs, by flow cytometric analysis. We found stronger binding of anti-CD34 mAbs to clonogenic, less differentiated, non-adherent neuroblastoma cells than to adherent neuroblastoma cells. Moreover, the majority of neuroblastoma cell lines shared hematopoietic-associated antigens with all blood cells. Because of these cross-reactions, especially found with the anti-CD34 mAbs 12.8 and ICH3, we have demonstrated that there is a potential risk of cell harvest contamination by circulating neuroblastoma cells during CD34⁺ stem cell selection.     

骨髓移植治疗Duchenne型肌营养不良症中骨髓细胞体内分布的实验研究

目的　探讨骨髓移植治疗肌营养不良症的早期骨髓细胞在体内的分布规律。方法　采用1 2 5I和99m Tc分别标记C5 7鼠的骨髓细胞和红细胞,将其静脉移植入放疗后的m dx鼠体内示踪,于4、12、2 4 h计算各器官内细胞特异性分布指数。用免疫荧光法对宿主骨骼肌的dystrophin检测。结果　骨髓细胞在移植后4 h以内随血流在体内均匀分布。在12 h时其在骨髓中的特异性分布指数明显增加(P<0 .0 5 ) ,而2 4 h时在病损骨骼肌中的特异性分布指数较正常骨骼肌明显增加(P<0 .0 5 ) ,并在2个月后宿主骨骼肌中表达了dystrophin。结论　骨髓移植后骨髓细胞有回巢现象和对病损骨骼肌有趋化现象,有利于靶器官的组织修复。该研究为骨髓移植后骨髓细胞早期能够定居在病损骨骼肌中并发挥修复功能提供了有力的实验依据。     

Stem cells for the treatment of spinal cord injury

This article reviews stem cell-based strategies for spinal cord injury repair, and practical issues concerning their translation to the clinic. Recent progress in the stem cell field includes clinically compliant culture conditions and directed differentiation of both embryonic stem cells and somatic stem cells. We provide a brief overview of the types of stem cells under evaluation, comparing their advantages and disadvantages for use in human clinical trials. We review the practical considerations and risks that must be addressed before human treatments can begin. With a growing understanding of these practical issues, stem cell biology, and spinal cord injury pathophysiology, stem cell-based therapies are moving closer to clinical application.     

5000/自体骨髓干细胞动员移植与手术移植治疗脊髓损伤的实验研究

探讨自体骨髓干细胞( bone marrow stem cells, BMSC)动员移植与手术移植治疗脊髓损伤（spinal cord injury，SCI）的疗效和机制。方法：选用10周龄健康SD大鼠90只，雌雄各半，建模前注射5-溴2-脱氧尿嘧啶核苷 (Bromodeoxyuridine,Brdu) 50mg/kg/d×3天后抽取自体骨髓，体外分离自体BMSC；NYU(New York University，NYU) Impactor制作SCI模型，随机分为对照组、动员移植组、手术移植组各30只。动员移植组应用重组粒细胞刺激因子（granulocyte-colony stimulating factor,G-CSF）皮下注射，20mg/kg/d×7天；手术移植组为损伤局部移植0.3ml（1×107个/ml）BMSC,各组均从术前三天开始，连续10天腹腔注射Brdu 50mg/kg/d。采用Basso-Beattie-Bresnahan（BBB）评分检测大鼠后肢的运动功能；体感诱发电位（somatoseneory evoked potential,SEP）和运动诱发电位（motor evoke potential,MEP）检测脊髓上、下行神经传导通路，判断SCI和恢复程度；病理和免疫组化观察脊髓损伤组织细胞结构变化及Brdu、GFAP和NSE分布表达。结果：BBB评分1周以后动员移植组和手术移植组分别较对照组比较差异有统计学意义（p＜0.05）,SEP、MEP潜伏期和波幅2周后动员移植组和手术移植组较对照组比较差异有统计学意义，组织病理学显示动员移植组和手术移植组较对照组有更少的空洞、坏死及GFAP阳性胶质瘢痕组织，较多的Brdu阳性细胞和NSE阳性细胞。结论：自体BMSCs动员移植和手术移植两种方法均能明显减轻SCI的程度，促进损伤后的脊髓功能的恢复，二者对比，前者更为方便、无创，实用性强，更有可能抓住有限的治疗时机，因而应用前景可能更好。     

骨髓基质细胞移植治疗脑缺血

脑缺血是临床上的常见病和多发病。现代药物及溶栓治疗的发展显示了对于急性期中风明显的功能保护作用；然而，神经损害一旦发生，对神经功能的恢复则显得治疗乏力。当前对于中风治疗的研究焦点集中在了干细胞，它不仅具有功能保护作用，而且使得细胞替代和功能恢复成为了可能。作为细胞治疗的种子细胞来源之一，骨髓基质细胞（BMSCs）分化潜能好，来源丰富，不存在胚胎干细胞移植所带来的伦理学问题。     

骨髓基质细胞立体定向移植治疗大鼠脑缺血损伤的实验研究

目的:观察骨髓基质细胞立体定向移植对大鼠脑缺血损伤后神经功能恢复的作用并探讨其作用机制。方法:制作SD大鼠大脑中动脉缺血模型(MCAO);体外培养骨髓基质细胞,观察其生物学特性以及立体定向移植后对脑缺血损伤后神经功能改善情况。结果:骨髓基质细胞体外可以长期传代、扩增,分泌NGF、VEGF等多种神经保护性因子;立体定向移植后,骨髓基质细胞在脑内存活、迁徙,小部分分化成具有神经元表面标志的细胞,与对照组相比,骨髓基质细胞移植组神经功能改善情况好于对照组。结论:骨髓基质细胞具有多向分化潜能,表达并分泌多种神经保护性营养因子。立体定向移植MSCs,对改善脑缺血损伤后的神经功能状况具有积极作用。     

Stem cell transplantation for treating stroke： status,trends and development

The developing approaches of thrombolytic therapy, endovascular treatment, neuroprotective therapy, and stem cell therapy have enabled breakthroughs in stroke treatment. In this study, we summarize and analyze trends and progress in stem cell transplantation for stroke treatment by retrieval of literature from Thomson Reuters Web of Science database, the NIH Clinical Trial Planning Grant Program, and Clinical Trials Registration Center in North America. In the last 10 years, there has been an increasing number of published articles on stem cell transplantation for stroke treatment. In particular, research from the USA and China has focused on stem cell transplantation. A total of 2,167 articles addressing stem cell transplantation for stroke treatment from 2004 to 2013 were retrieved from the Thomson Reuters Web of Science database. The ma- jority of these articles were from the USA （854, 39.4%）, with the journal Stroke publishing the most articles （145, 6.7%）. Of the published articles, 143 were funded by the National Institutes of Health （accounting for 6.6% of total publications）, and 91 by the National Natural Science Foundation of China. Between 2013 and 2014, the National Institutes of Health provided finan- cial support （$130 million subsidy） for 329 research projects on stroke therapy using stem cell transplantation. In 2014, 215 new projects were approved, receiving grants of up to $70,440,000. Ninety clinical trials focusing on stem cell transplantation for stroke were registered in the Clinical Trial Registration Center in North America, with 40 trials registered in the USA （ranked first place）. China had the maximum number of registered research or clinical trials （10 projects）.     

Autologous bone marrow mononuclear cell implantation for intracerebral hemorrhage—A prospective clinical observation

Background

This study was designed to assess the clinical effect of bone marrow mononuclear cells including mesenchymal stem cell (MSCs) in patients with intracerebral hemorrhage (ICH).

Methods

One hundred patients were divided into a study (n = 60) or a control group (n = 40). Bone marrow mononuclear cells from the same patient were injected to the perihemorrhage area in the base ganglia through an intracranial drainage tube 5.9 days after ICH. National Institute Stroke Scale (NIHSSS) and Barthel index was used to assess neurologic impairment and daily activities, respectively, before and 6 months after intervention.

Results

Six months after implantation, the NIHSS score in the study group was lower than in the control group (10.09 ± 8.86 vs 14.35 ± 10.14, P < 0.01), whereas the Barthel scores were higher (57.39 ± 23.51 vs 46.90 ± 20.29, P < 0.01). Neurological and functional improvement was observed in 52 (86.7%) of the study group patients, and in 17 (42.5%) of the control group patients (P = 0.001). No allergic or other adverse effects were observed in the study group.

Conclusion

Autologous bone marrow mononuclear cell implantation reduced neurological impairment and improved activities of daily living in a selected group of ICH patients. Further studies are required to ascertain the long-term safety and efficacy of this treatment.     

超顺磁性纳米铁粒子标记骨髓基质干细胞及对其分化能力的影响

目的研究超顺磁性纳米铁粒子标记骨髓基质干细胞(BMSCs)对其存活、增殖及向肝细胞分化能力的影响,确定最佳标记浓度,为磁共振成像(MRI)技术追踪同种异体移植的磁标记细胞奠定基础。方法使用菲立磁(Ferumoxides)标记大鼠BMSCs,采用Prussianblue染色、电镜等鉴定Ferumoxides标记BMSCs的效率;检测标记后BMSCs的诱导分化率评估其向肝细胞分化能力。门静脉和肝内局部移植标记BMSCs后行MRI肝脏扫描。结果标记培养基Fe3 浓度在11.2￣16.8μg/mL时,Prussianblue染色可见90%以上细胞阳性,透射电镜可见铁颗粒位于胞浆中;标记后BMSCs向肝细胞分化的能力与正常未标记BMSCs差异无统计学意义(P>0.05);Fe3 浓度在5.6μg/mL时,着色率<60%;Fe3 浓度为22.4μg/mL、28.0μg/mL时培养基中死细胞增多,且诱导分化后AFP与白蛋白阳性率降低,与对照组比较差异有统计学意义(P<0.05)。标记BMSCs门静脉和肝内局部移植后大鼠肝脏在MRI的SE-T2WI序列扫描下与移植前比较局部呈明显低信号改变。结论标记培养基Fe3 浓度在11.2￣16.8μg/mL时,Ferumoxides对BMSCs标记效率较高,且不影响其存活、增殖及向肝细胞分化能力,标记后的BMSCs可用于进一步实验。活体MRI示踪肝内或门静脉移植Ferumoxides标记的BMSCs有一定的可行性。     

大鼠骨髓间充质干细胞静脉移植对脊髓损伤的修复作用

目的初步探讨骨髓间充质干细胞（BMSCs）静脉移植对脊髓损伤后神经功能恢复和神经修复的影响。方法体外培养BMSCs，改良Allen法制备大鼠脊髓损伤模型，经尾静脉移植Brdu标记的BMSCs，损伤后24h、移植后1、3、5周评价实验动物的神经功能状况，并检测BMSCs在体内迁移、存活以及分化情况，电子显微镜观察组织形态学变化。结果移植的BMSCs在宿主损伤脊髓中聚集并存活，3～5周后有部分移植细胞表达神经元特异性烯醇化酶（NSE）、神经丝蛋白（NF）、微管相关蛋白（MAP2）；BMSCs静脉移植组大鼠运动功能改善，BBB评分高于对照组（P〈0．05）；5周后组织学观察，与对照组相比移植组损伤区脊髓结构较完整。结论BMSCs经静脉移植后可向脊髓损伤处聚集并存活分化，促进神经修复及神经功能的恢复。     

输注同基因骨髓间充质干细胞联合供体脾组织移植诱导肝移植大鼠免疫耐受的研究

摘要：目的 观察输注与受体同基因骨髓间充质干细胞联合供体脾组织移植对诱导大鼠肝移植术后免疫耐受作用,探讨其可能的机制。方法 实验鼠随机分为4组,每组24只。A组行DA-Lewis大鼠原位肝移植,B组行DA-Lewis大鼠原位肝移植术后予口服环孢素,C组行DA-Lewis大鼠原位肝移植同期输注Lewis大鼠骨髓间充质干细胞。D组在C组的基础上术中同期行DA大鼠脾组织移植。观察各组生存期,肝功能情况,血清细胞因子水平,嵌合体的形成情况及肝脏的病理变化。结果 与其他各组相比,D组大鼠存活时间明显延长,术后D组血清ALT、AST、TBIL较A组显著降低,与B、C组相近;B、C、D组IL-2、IFN－γ均升高,但低于A组（P<0.05）;、而IL-4和IL-10高于A组（P<0.05）,B、C组移植肝仅呈急性轻度排斥反应,A组呈急性重度排斥反应,D组未见明显排斥反应。30d后D组受体脾脏中供体阳性细胞明显高于各组（P<0.05）。结论 大鼠肝脏脾组织移植后输注与受体同基因骨髓间充质干细胞后能减轻移植肝的排斥作用,甚至诱导免疫耐受。     

骨髓基质干细胞在肌营养不良模型鼠中的分布及移植途径对分布的影响

目的研究干细胞在mdx鼠体内较长时间的动态分布以及移植途径对它的影响。方法用3H-TdR掺入标记骨髓基质干细胞,用4×106个/只静脉及腹腔移植放疗mdx鼠各30只,在24h、48h、2周、1个月、2个月、4个月时间点处死小鼠,取血、心、肝、脾、肺、肾、肌、脑、骨髓消化测定放射计数,计算%ID/g值。结果①静脉移植的mdx鼠脑、骨髓在2周才达到它自身的最高水平,心肌、骨骼肌4个月时达到高峰;而每个时间点各器官含量相比早期基本是肺、肝、骨髓分布多,心肌、肌肉2个月后仅次于骨髓。②腹腔移植mdx鼠血、肺、肾、肝、脾脏、脑到达它各自的高峰比静脉注射均有不同时间延迟,早期还是在骨髓、肝、肺分布多,肌肉2个月后仅次于骨髓。③心、肌肉、脑、骨髓含量静脉移植比腹腔移植高,均具有显著的统计学意义(P<0.01)。结论干细胞移植mdx鼠后4个月达高峰,移植途径对干细胞的分布有影响,3H-TdR能长时间动态观察干细胞分布。     

深低温冻储同种异体气管移植并骨髓间充质干细胞移植后气管上皮细胞血管内皮生长因子的表达

背景：深低温冷冻后的组织可以保持其原有的活力和组织完整性,并能消减抗原性。目的：静脉移植骨髓间充质干细胞合并深低温处理供体气管后进行同种异体气管移植,观察骨髓间充质干细胞在促进上皮再生和再血管化后的作用。方法：用深低温冻储2 周和6 周的气管进行Wistar大鼠同种异体气管移植后,用PKH-26标记的3~5代骨髓间充质干细胞经鼠尾静脉移植入受体内,等量的磷酸盐缓冲液注射作为实验对照。观察供体气管的组织学和血管内皮生长因子蛋白表达。结果和结论：骨髓间充质干细胞移植合并深低温冻储后的移植气管结构完整,软骨无变性坏死;深低温6 周的移植气管上皮再生情况优于2 周。骨髓间充质干细胞移植组的上皮细胞表达血管内皮生长因子水平高于磷酸盐缓冲液对照组。结果提示,骨髓间充质干细胞移植合并深低温冻储6 周后移植气管存活期好于深低温冻储2周;骨髓间充质干细胞能够促进移植物周围新生血管的增加,从而促进气管损伤的修复。