CXCR4基因沉默和SDF-1α对骨髓间质干细胞脑内迁移的影响
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摘要
缺血性脑卒中是中老年人致死致残的主要疾病之一,目前尚无有效药物能够阻止脑缺血和再灌注损伤引起的多重神经损害。许多动物实验表明,骨髓间质干细胞(MSCs)经全身或局部移植后能促进卒中后神经功能的修复。在大鼠脑卒中和创伤模型中,经静脉或大脑内直接注射MSCs后,MSCs可穿过血脑屏障向损伤脑组织聚集,进而修复神经功能。其中,损伤组织似乎可特异吸引MSCs向损伤区迁移,然而,目前对调节MSCs迁移进而向损伤脑组织聚集的机制还不清楚。
已知基质细胞衍生因子-1α(SDF-1α)和其唯一特异受体CXCR4与干细胞定向迁移和损伤修复密切相关。在大鼠心肌梗死模型中,SDF-1在促使表达CXCR4的MSCs向缺血心肌的定向迁移、聚集中起很重要作用。而SDF-1α/CXCR4在低氧条件下表达增加,如急性肾衰、缺血性心脏病、脑缺血。为此,推测SDF-1α/CXCR4在MSCs向缺血脑组织的定向迁移中可能也起很重要作用。
RNA干扰(RNAi)技术可稳定沉默哺乳动物细胞内的靶向基因,是研究基因功能的强大工具。为探讨脑缺血损伤中MSCs的迁移和调节机制,我们首先构建CXCR4 RNAi慢病毒载体,进而建立CXCR4基因稳定沉默的大鼠MSCs(rMSCs)。随后通过体内、外实验研究CXCR4基因沉默和SDF-1α对rMSCs迁移的影响。本课题分三部分。第一部分:CXCR4基因RNAi慢病毒载体的构建及鉴定
1、材料和方法
1)根据大鼠CXCR4 mRNA序列,选择三个21nts的靶序列,设计并合成包含各正、反义靶序列的互补DNA链;
2)退火后插入pSUPER载体的H1 RNA启动子后面,产生pRiCXCR4a, b, c;
3)酶切电泳和测序鉴定;
4)质粒鉴定正确后,将其中的CXCR4 shRNA表达结构酶切,插入到慢病毒载体质粒pNL中,产生pNL-RiCXCR4a, b, c。
2、结果
1)酶切鉴定和测序都证实pRiCXCR4质粒构建正确。
2)将pRiCXCR4中的CXCR4 shRNA表达结构插入pNL质粒,产生能同时表达增强型绿色荧光蛋白(EGFP)和转录产生CXCR4 shRNA的慢病毒载体质粒,测序鉴定正确。
3、结论
1)成功构建大鼠CXCR4 shRNA表达载体pRiCXCR4。
2)成功构建大鼠CXCR4基因RNAi慢病毒载体pNL-RiCXCR4。
3)为通过阻断大鼠CXCR4基因表达进而研究CXCR4功能奠定了基础。
第二部分:CXCR4基因沉默的骨髓间质干细胞的构建、培养与鉴定
1、材料和方法
1)将三质粒pNL-RiCXCR4、pHELPER和pVSVG共转染293T细胞,包装生产慢病毒;
2)经超速离心收集病毒颗粒,转导293T细胞,用流式细胞术测定EGFP蛋白表达率来测定慢病毒功能滴度;
3)用相同滴度的慢病毒转导rMSCs ,建立CXCR4基因稳定沉默细胞系rMSCs/pNL-RiCXCR4(MSC-CXCR4-);
4)用实时定量RT-PCR、Western Blot和流式细胞术检测CXCR4 mRNA、蛋白质和细胞表面表达水平,筛选有效的CXCR4基因沉默的细胞系MSC-CXCR4-;
5)绘制细胞生长曲线,计算细胞倍增时间,比较CXCR4基因沉默的rMSCs与亲本细胞的生长变化。
2、结果
1)三质粒pNL-RiCXCR4、pHELPER和pVSVG共转染293T细胞,包装产生慢病毒。
2)未浓缩含病毒细胞培养上清和浓缩病毒悬液的功能滴度分别为6.4×104TU/mL和6.9×106TU/mL。
3) CXCR4基因稳定沉默细胞MSC-CXCR4-强烈表达EGFP。
4)实时定量RT-PCR、Western Blot和流式细胞术一致证实pNL-RiCXCR4b组的CXCR4抑制作用最显著。
5)亲本细胞、非特异细胞和CXCR4基因沉默细胞的生长曲线和倍增时间无明显差异。
3、结论
1)包装产生高滴度慢病毒,并能高效转导rMSCs。
2)建立CXCR4基因稳定沉默细胞系MSC-CXCR4-。
3)实时定量RT-PCR分析CXCR4 mRNA、Western Blot检测CXCR4蛋白和流式细胞术检测rMSCs表面CXCR4抗原,均证实所构建的慢病毒载体能使rMSCs的CXCR4基因表达显著下调。
4)细胞生长曲线测定实验证明,CXCR4基因沉默rMSCs的生长未受到明显影响。第三部分:CXCR4基因沉默及SDF-1α对骨髓间质干细胞迁移作用的体内、外实验研究1、材料和方法1)体外实验在内置8μm孔径聚碳酸酯膜的48孔微迁移板中进行,趋化因子SDF-1α以不同浓度加入到迁移板下室,细胞rMSCs或CXCR4基因沉默的rMSCs加入到上室,观察细胞迁移情况,计算细胞迁移指数。
2)取健康成年雄性SD大鼠制作短暂大脑中动脉阻塞(MCAO)模型。在MCAO前24h或MCAO后24h将表达EGFP(EGFP+)的rMSCs经股静脉移植入大鼠体内。其中,EGFP+的CXCR4基因沉默的rMSCs仅移植入MCAO后24h的大鼠体内。
3)将EGFP阳性(EGFP+)的rMSCs经股静脉移植入正常大鼠体内后,立即将SDF-1α直接注射入左侧大脑皮质。作为对照,对侧相应部位只注射PBS。
4)取大鼠骨髓制作涂片,同时取脑、心、肺、肝、脾、肾、骨骼肌、小肠组织标本制作冰冻切片,在荧光显微镜下观察EGFP+细胞的分布情况。
5)为明确脑组织中SDF-1α表达和EGFP+细胞迁移之间的定位关系,对脑组织冰冻切片进行SDF-1α的免疫荧光染色。
2、结果
1)体外微迁移板实验表明,加入200ng/mL SDF-1α后可明显促进rMSCs的跨膜迁移,而rMSCs中CXCR4基因沉默后,rMSCs的跨膜迁移明显减弱。
2)对MCAO前大鼠或对未进行MCAO的正常大鼠移植EGFP+细胞后,可在其肺、骨髓、脾、小肠发现EGFP+细胞,而在其他组织未发现。
3)对MCAO后大鼠移植EGFP+细胞后,发现大量EGFP+细胞聚集于大脑半球缺血半暗带区,在肺中也可见EGFP+细胞分布,在缺血对侧大脑半球及其他组织中未发现。而rMSCs中CXCR4基因沉默后,这种EGFP+细胞在大脑半球缺血区的聚集作用被明显抑制。
4)免疫荧光染色显示,在脑组织切片中SDF-1α主要由缺血半暗带区神经元和星形胶质细胞分泌,EGFP+细胞的数量和定位与SDF-1α的表达量大体一致。
3、结论
1)移植前的脑组织急性损伤对移植的rMSCs向脑局部缺血损伤区定向迁移起决定性作用,否则移植细胞将在骨髓和脾脏较长时间停留且不易再次迁移。
2)损伤脑组织局部产生的SDF-1α和表达于rMSCs表面的CXCR4在介导rMSCs的损伤组织定向迁移中起很重要作用,在MSCs移植治疗脑梗死中,我们可能可通过调节SDF-1α/CXCR4表达以达到增进治疗作用的目的。
Stroke is a leading cause of death and no pharmacological treatment is presently available to protect brain tissue from the multiple neurochemical cascades that are triggered by ischemia and reperfusion. Mounting experimental data obtained from animal models suggest that bone marrow mesenchymal stem cells (MSCs), administered locally or systemically, can improve functional outcome after stroke. In rat models for stroke and trauma, intravenously and transcerebrally injected MSCs have been demonstrated to go through the blood-brain barrier and accumulate in the parenchyma of the injured brain, and thereby induce neurological and functional recovery. It therefore seems that the injured tissues can specifically attract MSCs and mediate their migration behavior. However, the mechanisms regulating MSCs migration and accumulation in the injured brain remain to be revealed.
Stromal cell-derived factor-1 (SDF-1α) and its cellular receptor CXCR4 have been demonstrated to direct the migration of stem cells associated with injury repair in many species and tissue types. In rat myocardial infarction model, SDF-1 plays an essential role in promoting the homing in of cells toward the ischemic myocardium by recruiting MSCs that express CXCR4. Expression of both SDF-1α/CXCR4 is predominantly promoted in hypoxic conditions, such as acute renal failure, ischemic cardiomyopathy, and ischemic brain. This raises the possibility that the SDF-1α/CXCR4 axis also plays essential roles in directing MSCs migration in ischemic brain.
RNA interference (RNAi) technology, silencing targeted genes in mammalian cells, has become a powerful tool for studying gene function. In an effort to investigate the migration of rat MSCs (rMSCs) within the injured brain and the regulating mechanisms, we first constructed CXCR4 RNAi lentiviral vector and established CXCR4 gene silencing rMSCs. Then we focused on characterizing the effect of CXCR4 gene silencing and SDF-1αon the migration of rMSCs in vitro and vivo. This study consisted of three parts.
Part1:Construction and identification of the lentiviral RNAi vector of CXCR4 gene Materials and Methods
1) Three specific target sequences were selected according to rat CXCR4 mRNA. The complementary DNA which contained both sense and antisense oligonucleotides were synthesized.
2) After phosphorylation and annealing, these double strands DNA were cloned to BglⅡand HindⅢsites of pSUPER.
3) Then the product pRiCXCR4a, b, c was confirmed by electrophoresis and sequencing.
4) CXCR4 shRNA was cloned to XbaI site of pNL, a transfer vector of lentivirus. Then the product pNL-RiCXCR4a, b, c were obtained.
Results and Conclusions
1) It is confirmed by restriction enzyme digestion and sequencing that CXCR4 shRNA expression structure is correctly cloned to pSUPER and pNL respectively.
2) RNAi lentivirus vector of rat CXCR4 gene has been constructed, which is the essential building block required for the CXCR4 functional research. Part2:Establishment, culture and identification of CXCR4 gene silenced MSCs
Materials and Methods
1) pNL-RiCXCR4 was cotransfected along with pHELPER and pVSVG into 293T to package lentivirus particles.
2) According to the enhanced green fluorescent protein (EGFP) expression,the functional titer was determined by flow cytometry (FCM) after transduction into 293T cells.
3) The rMSCs were transduced with lentiviral vectors at 2MOI and selected for stable integrants by using EGFP reporter gene to establish stable silenced cell line.
4) Real-time RT-PCR analysis of CXCR4 mRNA, Western Blot analysis of CXCR4 protein and FCM analysis of CXCR4 on the cell membrane were conducted to compare the RNAi effect.
5) The growth curve was obtained and the population doubling time was calculated to compare the growth of CXCR4-downregulated rMSCs and their parental cells.
Results and Conclusions
1) After cotransfection, lentiviral vector can be packaged in 293T cells.
2) The functional titer of unconcentrated virus and concentrated virus are 6.4×104TU/mL and 6.9×106TU/mL respectively.
3) CXCR4 silenced cell line MSC-CXCR4- was successfully established.
4) Down-regulation of CXCR4 expression in rMSCs was confirmed by Real-time RT-PCR, Western blot and FCM.
Part3:Effect of CXCR4 gene silencing and SDF-1αon the migration of rMSCs in vitro and vivo
Materials and Methods
1) In vitro migration of rMSCs in response to SDF-1αwas assessed in a modified 48-well microchemotaxis chamber using polycarbonate membranes with 8μm pore size. The cells were added to the upper chambers. SDF-1αwas added to the lower wells in different concentrations. The migrated cells were counted and the migration index was calculated to show the difference of migration.
2) The healthy adult male SD rats were subjected to transient middle cerebral artery occlusion (MCAO).The rMSCs expressed EGFP (EGFP+) were infused into the femoral vein of rats at 24 hours before or after MCAO. The rMSCs with CXCR4 gene downregulation were also transplanted at 24 hours after MCAO.
3) Immediately following femoral vein injection of EGFP-labeled rMSCs, the SD rats received an intracerebral injection of SDF-1αinto the left cerebral cortex. As control, the opposite side was injected with PBS alone.
4) The tissue samples of brain, heart, lung, liver, spleen, kidney, skeletal muscle, small intestine and bone marrow of rats were collected. Then the frozen sections and bone marrow smear were prepared. The distribution of EGFP+ cells was observed under the fluorescent microscope.
5) To clarify the relation and location of SDF-1αand EGFP-labeled rMSCs in the brain, SDF-1αimmunofluorescence staining was performed in the brain frozen sections.
Results and Conclusions
1) In vitro chemotaxis assay, the number of migrating cells significantly increased in the concentration of 200ng/mL SDF-1α, and significantly decreased while CXCR4 was knockdown with siRNA.
2) In the groups of rats transplanted before or without MCAO, EGFP+ cells were found in bone marrow sample, lung, spleen and intestine samples, but not in the other tissues.
3) In the group of rats transplanted after MCAO, EGFP+ cells were localized in the ischemic penumbra, few marked cells were observed in the contralateral cerebral hemisphere or other tissues except a few in lung. CXCR4 downregulation in rMSCs exhibit strong inhibitory effects on rMSCs migration to the ischemic region of brain.
4) Immunofluorescence staining showed that the source of SDF-1αis mainly from neurons and astrocytes in the ischemic penumbra, and the location and density of EGFP+ cells appears to be roughly associated with the level of SDF-1αimmunoreactivity in the brain frozen sections.
5) The cerebral injury before transplantation is very important and necessary for intravenously grafted rMSCs selectively migrating to ischemic focus, otherwise, the grafted rMSCs will settle down in bone marrow, spleen, and never migrate to other tissues again.
6) The interaction of locally produced SDF-1αand CXCR4 expressed on the rMSCs surface plays an important role in the migration of transplanted cells, suggesting that it might be a potential approach to modulate the expression of the two molecules in order to further facilitate the therapeutic effects using MSCs.
已知基质细胞衍生因子-1α(SDF-1α)和其唯一特异受体CXCR4与干细胞定向迁移和损伤修复密切相关。在大鼠心肌梗死模型中,SDF-1在促使表达CXCR4的MSCs向缺血心肌的定向迁移、聚集中起很重要作用。而SDF-1α/CXCR4在低氧条件下表达增加,如急性肾衰、缺血性心脏病、脑缺血。为此,推测SDF-1α/CXCR4在MSCs向缺血脑组织的定向迁移中可能也起很重要作用。
RNA干扰(RNAi)技术可稳定沉默哺乳动物细胞内的靶向基因,是研究基因功能的强大工具。为探讨脑缺血损伤中MSCs的迁移和调节机制,我们首先构建CXCR4 RNAi慢病毒载体,进而建立CXCR4基因稳定沉默的大鼠MSCs(rMSCs)。随后通过体内、外实验研究CXCR4基因沉默和SDF-1α对rMSCs迁移的影响。本课题分三部分。第一部分:CXCR4基因RNAi慢病毒载体的构建及鉴定
1、材料和方法
1)根据大鼠CXCR4 mRNA序列,选择三个21nts的靶序列,设计并合成包含各正、反义靶序列的互补DNA链;
2)退火后插入pSUPER载体的H1 RNA启动子后面,产生pRiCXCR4a, b, c;
3)酶切电泳和测序鉴定;
4)质粒鉴定正确后,将其中的CXCR4 shRNA表达结构酶切,插入到慢病毒载体质粒pNL中,产生pNL-RiCXCR4a, b, c。
2、结果
1)酶切鉴定和测序都证实pRiCXCR4质粒构建正确。
2)将pRiCXCR4中的CXCR4 shRNA表达结构插入pNL质粒,产生能同时表达增强型绿色荧光蛋白(EGFP)和转录产生CXCR4 shRNA的慢病毒载体质粒,测序鉴定正确。
3、结论
1)成功构建大鼠CXCR4 shRNA表达载体pRiCXCR4。
2)成功构建大鼠CXCR4基因RNAi慢病毒载体pNL-RiCXCR4。
3)为通过阻断大鼠CXCR4基因表达进而研究CXCR4功能奠定了基础。
第二部分:CXCR4基因沉默的骨髓间质干细胞的构建、培养与鉴定
1、材料和方法
1)将三质粒pNL-RiCXCR4、pHELPER和pVSVG共转染293T细胞,包装生产慢病毒;
2)经超速离心收集病毒颗粒,转导293T细胞,用流式细胞术测定EGFP蛋白表达率来测定慢病毒功能滴度;
3)用相同滴度的慢病毒转导rMSCs ,建立CXCR4基因稳定沉默细胞系rMSCs/pNL-RiCXCR4(MSC-CXCR4-);
4)用实时定量RT-PCR、Western Blot和流式细胞术检测CXCR4 mRNA、蛋白质和细胞表面表达水平,筛选有效的CXCR4基因沉默的细胞系MSC-CXCR4-;
5)绘制细胞生长曲线,计算细胞倍增时间,比较CXCR4基因沉默的rMSCs与亲本细胞的生长变化。
2、结果
1)三质粒pNL-RiCXCR4、pHELPER和pVSVG共转染293T细胞,包装产生慢病毒。
2)未浓缩含病毒细胞培养上清和浓缩病毒悬液的功能滴度分别为6.4×104TU/mL和6.9×106TU/mL。
3) CXCR4基因稳定沉默细胞MSC-CXCR4-强烈表达EGFP。
4)实时定量RT-PCR、Western Blot和流式细胞术一致证实pNL-RiCXCR4b组的CXCR4抑制作用最显著。
5)亲本细胞、非特异细胞和CXCR4基因沉默细胞的生长曲线和倍增时间无明显差异。
3、结论
1)包装产生高滴度慢病毒,并能高效转导rMSCs。
2)建立CXCR4基因稳定沉默细胞系MSC-CXCR4-。
3)实时定量RT-PCR分析CXCR4 mRNA、Western Blot检测CXCR4蛋白和流式细胞术检测rMSCs表面CXCR4抗原,均证实所构建的慢病毒载体能使rMSCs的CXCR4基因表达显著下调。
4)细胞生长曲线测定实验证明,CXCR4基因沉默rMSCs的生长未受到明显影响。第三部分:CXCR4基因沉默及SDF-1α对骨髓间质干细胞迁移作用的体内、外实验研究1、材料和方法1)体外实验在内置8μm孔径聚碳酸酯膜的48孔微迁移板中进行,趋化因子SDF-1α以不同浓度加入到迁移板下室,细胞rMSCs或CXCR4基因沉默的rMSCs加入到上室,观察细胞迁移情况,计算细胞迁移指数。
2)取健康成年雄性SD大鼠制作短暂大脑中动脉阻塞(MCAO)模型。在MCAO前24h或MCAO后24h将表达EGFP(EGFP+)的rMSCs经股静脉移植入大鼠体内。其中,EGFP+的CXCR4基因沉默的rMSCs仅移植入MCAO后24h的大鼠体内。
3)将EGFP阳性(EGFP+)的rMSCs经股静脉移植入正常大鼠体内后,立即将SDF-1α直接注射入左侧大脑皮质。作为对照,对侧相应部位只注射PBS。
4)取大鼠骨髓制作涂片,同时取脑、心、肺、肝、脾、肾、骨骼肌、小肠组织标本制作冰冻切片,在荧光显微镜下观察EGFP+细胞的分布情况。
5)为明确脑组织中SDF-1α表达和EGFP+细胞迁移之间的定位关系,对脑组织冰冻切片进行SDF-1α的免疫荧光染色。
2、结果
1)体外微迁移板实验表明,加入200ng/mL SDF-1α后可明显促进rMSCs的跨膜迁移,而rMSCs中CXCR4基因沉默后,rMSCs的跨膜迁移明显减弱。
2)对MCAO前大鼠或对未进行MCAO的正常大鼠移植EGFP+细胞后,可在其肺、骨髓、脾、小肠发现EGFP+细胞,而在其他组织未发现。
3)对MCAO后大鼠移植EGFP+细胞后,发现大量EGFP+细胞聚集于大脑半球缺血半暗带区,在肺中也可见EGFP+细胞分布,在缺血对侧大脑半球及其他组织中未发现。而rMSCs中CXCR4基因沉默后,这种EGFP+细胞在大脑半球缺血区的聚集作用被明显抑制。
4)免疫荧光染色显示,在脑组织切片中SDF-1α主要由缺血半暗带区神经元和星形胶质细胞分泌,EGFP+细胞的数量和定位与SDF-1α的表达量大体一致。
3、结论
1)移植前的脑组织急性损伤对移植的rMSCs向脑局部缺血损伤区定向迁移起决定性作用,否则移植细胞将在骨髓和脾脏较长时间停留且不易再次迁移。
2)损伤脑组织局部产生的SDF-1α和表达于rMSCs表面的CXCR4在介导rMSCs的损伤组织定向迁移中起很重要作用,在MSCs移植治疗脑梗死中,我们可能可通过调节SDF-1α/CXCR4表达以达到增进治疗作用的目的。
Stroke is a leading cause of death and no pharmacological treatment is presently available to protect brain tissue from the multiple neurochemical cascades that are triggered by ischemia and reperfusion. Mounting experimental data obtained from animal models suggest that bone marrow mesenchymal stem cells (MSCs), administered locally or systemically, can improve functional outcome after stroke. In rat models for stroke and trauma, intravenously and transcerebrally injected MSCs have been demonstrated to go through the blood-brain barrier and accumulate in the parenchyma of the injured brain, and thereby induce neurological and functional recovery. It therefore seems that the injured tissues can specifically attract MSCs and mediate their migration behavior. However, the mechanisms regulating MSCs migration and accumulation in the injured brain remain to be revealed.
Stromal cell-derived factor-1 (SDF-1α) and its cellular receptor CXCR4 have been demonstrated to direct the migration of stem cells associated with injury repair in many species and tissue types. In rat myocardial infarction model, SDF-1 plays an essential role in promoting the homing in of cells toward the ischemic myocardium by recruiting MSCs that express CXCR4. Expression of both SDF-1α/CXCR4 is predominantly promoted in hypoxic conditions, such as acute renal failure, ischemic cardiomyopathy, and ischemic brain. This raises the possibility that the SDF-1α/CXCR4 axis also plays essential roles in directing MSCs migration in ischemic brain.
RNA interference (RNAi) technology, silencing targeted genes in mammalian cells, has become a powerful tool for studying gene function. In an effort to investigate the migration of rat MSCs (rMSCs) within the injured brain and the regulating mechanisms, we first constructed CXCR4 RNAi lentiviral vector and established CXCR4 gene silencing rMSCs. Then we focused on characterizing the effect of CXCR4 gene silencing and SDF-1αon the migration of rMSCs in vitro and vivo. This study consisted of three parts.
Part1:Construction and identification of the lentiviral RNAi vector of CXCR4 gene Materials and Methods
1) Three specific target sequences were selected according to rat CXCR4 mRNA. The complementary DNA which contained both sense and antisense oligonucleotides were synthesized.
2) After phosphorylation and annealing, these double strands DNA were cloned to BglⅡand HindⅢsites of pSUPER.
3) Then the product pRiCXCR4a, b, c was confirmed by electrophoresis and sequencing.
4) CXCR4 shRNA was cloned to XbaI site of pNL, a transfer vector of lentivirus. Then the product pNL-RiCXCR4a, b, c were obtained.
Results and Conclusions
1) It is confirmed by restriction enzyme digestion and sequencing that CXCR4 shRNA expression structure is correctly cloned to pSUPER and pNL respectively.
2) RNAi lentivirus vector of rat CXCR4 gene has been constructed, which is the essential building block required for the CXCR4 functional research. Part2:Establishment, culture and identification of CXCR4 gene silenced MSCs
Materials and Methods
1) pNL-RiCXCR4 was cotransfected along with pHELPER and pVSVG into 293T to package lentivirus particles.
2) According to the enhanced green fluorescent protein (EGFP) expression,the functional titer was determined by flow cytometry (FCM) after transduction into 293T cells.
3) The rMSCs were transduced with lentiviral vectors at 2MOI and selected for stable integrants by using EGFP reporter gene to establish stable silenced cell line.
4) Real-time RT-PCR analysis of CXCR4 mRNA, Western Blot analysis of CXCR4 protein and FCM analysis of CXCR4 on the cell membrane were conducted to compare the RNAi effect.
5) The growth curve was obtained and the population doubling time was calculated to compare the growth of CXCR4-downregulated rMSCs and their parental cells.
Results and Conclusions
1) After cotransfection, lentiviral vector can be packaged in 293T cells.
2) The functional titer of unconcentrated virus and concentrated virus are 6.4×104TU/mL and 6.9×106TU/mL respectively.
3) CXCR4 silenced cell line MSC-CXCR4- was successfully established.
4) Down-regulation of CXCR4 expression in rMSCs was confirmed by Real-time RT-PCR, Western blot and FCM.
Part3:Effect of CXCR4 gene silencing and SDF-1αon the migration of rMSCs in vitro and vivo
Materials and Methods
1) In vitro migration of rMSCs in response to SDF-1αwas assessed in a modified 48-well microchemotaxis chamber using polycarbonate membranes with 8μm pore size. The cells were added to the upper chambers. SDF-1αwas added to the lower wells in different concentrations. The migrated cells were counted and the migration index was calculated to show the difference of migration.
2) The healthy adult male SD rats were subjected to transient middle cerebral artery occlusion (MCAO).The rMSCs expressed EGFP (EGFP+) were infused into the femoral vein of rats at 24 hours before or after MCAO. The rMSCs with CXCR4 gene downregulation were also transplanted at 24 hours after MCAO.
3) Immediately following femoral vein injection of EGFP-labeled rMSCs, the SD rats received an intracerebral injection of SDF-1αinto the left cerebral cortex. As control, the opposite side was injected with PBS alone.
4) The tissue samples of brain, heart, lung, liver, spleen, kidney, skeletal muscle, small intestine and bone marrow of rats were collected. Then the frozen sections and bone marrow smear were prepared. The distribution of EGFP+ cells was observed under the fluorescent microscope.
5) To clarify the relation and location of SDF-1αand EGFP-labeled rMSCs in the brain, SDF-1αimmunofluorescence staining was performed in the brain frozen sections.
Results and Conclusions
1) In vitro chemotaxis assay, the number of migrating cells significantly increased in the concentration of 200ng/mL SDF-1α, and significantly decreased while CXCR4 was knockdown with siRNA.
2) In the groups of rats transplanted before or without MCAO, EGFP+ cells were found in bone marrow sample, lung, spleen and intestine samples, but not in the other tissues.
3) In the group of rats transplanted after MCAO, EGFP+ cells were localized in the ischemic penumbra, few marked cells were observed in the contralateral cerebral hemisphere or other tissues except a few in lung. CXCR4 downregulation in rMSCs exhibit strong inhibitory effects on rMSCs migration to the ischemic region of brain.
4) Immunofluorescence staining showed that the source of SDF-1αis mainly from neurons and astrocytes in the ischemic penumbra, and the location and density of EGFP+ cells appears to be roughly associated with the level of SDF-1αimmunoreactivity in the brain frozen sections.
5) The cerebral injury before transplantation is very important and necessary for intravenously grafted rMSCs selectively migrating to ischemic focus, otherwise, the grafted rMSCs will settle down in bone marrow, spleen, and never migrate to other tissues again.
6) The interaction of locally produced SDF-1αand CXCR4 expressed on the rMSCs surface plays an important role in the migration of transplanted cells, suggesting that it might be a potential approach to modulate the expression of the two molecules in order to further facilitate the therapeutic effects using MSCs.
引文
1 Picinich SC, Mishra PJ, Mishra PJ, et al. The therapeutic potential of mesenchymal stem cells. Cell- & tissue-based therapy. Expert Opin Biol Ther, 2007,7:965-973.
2 Fox JM, Chamberlain G, Ashton BA, et al. Recent advances into the understanding of mesenchymal stem cell trafficking. Br J Haematol, 2007,137:491-502.
3 Brooke G, Cook M, Blair C, et al. Therapeutic applications of mesenchymal stromal cells. Semin Cell Dev Biol, 2007,18:846-858.
4 Shyu WC, Lee YJ, Liu DD, et al. Homing genes, cell therapy and stroke. Front Biosci, 2006,11:899-907.
5 Chen J, Li Y, Wang L, et al. Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats. J Neurol Sci JT - Journal of the neurological sciences, 2001,189:49-57.
6 Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke JT - Stroke; a journal of cerebral circulation, 2001,32:1005-1011.
7 Lu D, Mahmood A, Wang L, et al. Adult bone marrow stromal cells administered intravenously to rats after traumatic brain injury migrate into brain and improve neurological outcome. Neuroreport JT - Neuroreport, 2001,12:559-563.
8 Lee RH, Hsu SC, Munoz J, et al. A subset of human rapidly self-renewing marrow stromal cells preferentially engraft in mice. Blood, 2006,107:2153-2161.
9 Kim CH, Broxmeyer HE. Chemokines: signal lamps for trafficking of T and B cells for development and effector function. J Leukoc Biol, 1999,65:6-15.
10 Hattori K, Heissig B, Rafii S. The regulation of hematopoietic stem cell and progenitor mobilization by chemokine SDF-1. Leuk Lymphoma JT - Leukemia & lymphoma, 2003,44:575-582.
11 Salvucci O, Yao L, Villalba S, et al. Regulation of endothelial cell branching morphogenesis by endogenous chemokine stromal-derived factor-1. Blood JT - Blood, 2002,99:2703-2711.
12 Yamaguchi J, Kusano KF, Masuo O, et al. Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization. Circulation JT - Circulation, 2003,107:1322-1328.
13 Tham TN, Lazarini F, Franceschini IA, et al. Developmental pattern of expression of the alpha chemokine stromal cell-derived factor 1 in the rat central nervous system. Eur J Neurosci JT - The European journal of neuroscience, 2001,13:845-856.
14 Petit I, Szyper-Kravitz M, Nagler A, et al. G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol JT - Nature immunology, 2002,3:687-694.
15 Stumm RK, Rummel J, Junker V, et al. A dual role for the SDF-1/CXCR4 chemokine receptor system in adult brain: isoform-selective regulation of SDF-1 expression modulates CXCR4-dependent neuronal plasticity and cerebral leukocyte recruitment after focal ischemia. J Neurosci JT - The Journal of neuroscience : the official journal of the Society for Neuroscience, 2002,22:5865-5878.
16 Askari AT, Unzek S, Popovic ZB, et al. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet JT - Lancet, 2003,362:697-703.
17 Downward J. RNA interference. BMJ, 2004,328:1245-1248.
18 Scherr M, Morgan MA, Eder M. Gene silencing mediated by small interfering RNAs in mammalian cells. Curr Med Chem, 2003,10:245-256.
19 Rubinson DA, Dillon CP, Kwiatkowski AV, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet, 2003,33:401-406.
20 Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA, 2003,9:493-501.
21 Baggiolini M. Chemokines and leukocyte traffic. Nature, 1998,392:565-568.
22 Melchers F, Rolink AG, Schaniel C. The role of chemokines in regulating cell migration during humoral immune responses. Cell, 1999,99:351-354.
23 Murdoch C, Finn A. Chemokine receptors and their role in inflammation and infectious diseases. Blood, 2000,95:3032-3043.
24 Bazan JF, Bacon KB, Hardiman G, et al. A new class of membrane-bound chemokine with a CX3C motif. Nature, 1997,385:640-644.
25 Kelner GS, Kennedy J, Bacon KB, et al. Lymphotactin: a cytokine that represents a new class of chemokine. Science, 1994,266:1395-1399.
26 Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity, 2000,12:121-127.
27 Wang L, Li Y, Chen X, et al. MCP-1, MIP-1, IL-8 and ischemic cerebral tissue enhance human bone marrow stromal cell migration in interface culture. Hematology, 2002,7:113-117.
28 Ji JF, He BP, Dheen ST, et al. Interactions of chemokines and chemokine receptors mediate the migration of mesenchymal stem cells to the impaired site in the brain afterhypoglossal nerve injury. Stem Cells, 2004,22:415-427.
29 Wang Y, Deng Y, Zhou GQ. SDF-1alpha/CXCR4-mediated migration of systemically transplanted bone marrow stromal cells towards ischemic brain lesion in a rat model. Brain Res, 2008,1195:104-112.
30 Hill WD, Hess DC, Martin-Studdard A, et al. SDF-1 (CXCL12) is upregulated in the ischemic penumbra following stroke: association with bone marrow cell homing to injury. J Neuropathol Exp Neurol, 2004,63:84-96.
31 Cui X, Chen J, Zacharek A, et al. Nitric oxide donor upregulation of stromal cell-derived factor-1/chemokine (CXC motif) receptor 4 enhances bone marrow stromal cell migration into ischemic brain after stroke. Stem Cells, 2007,25:2777-2785.
32 Shirozu M, Nakano T, Inazawa J, et al. Structure and chromosomal localization of the human stromal cell-derived factor 1 (SDF1) gene. Genomics, 1995,28:495-500.
33 Loetscher M, Geiser T, O'Reilly T, et al. Cloning of a human seven-transmembrane domain receptor, LESTR, that is highly expressed in leukocytes. J Biol Chem, 1994,269:232-237.
34 Feng Y, Broder CC, Kennedy PE, et al. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science, 1996,272:872-877.
35 Horuk R. Chemokines beyond inflammation. Nature, 1998,393:524-525.
36 Horuk R. Chemokine receptors. Cytokine Growth Factor Rev, 2001,12:313-335.
37 Muller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature, 2001,410:50-56.
38 Scotton CJ, Wilson JL, Scott K, et al. Multiple actions of the chemokine CXCL12 on epithelial tumor cells in human ovarian cancer. Cancer Res, 2002,62:5930-5938.
39 Hideshima T, Chauhan D, Hayashi T, et al. The biological sequelae of stromal cell-derived factor-1alpha in multiple myeloma. Mol Cancer Ther, 2002,1:539-544.
40 Taichman RS, Cooper C, Keller ET, et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone. Cancer Res, 2002,62:1832-1837.
41 Peled A, Petit I, Kollet O, et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science, 1999,283:845-848.
42 Hesselgesser J, Halks-Miller M, DelVecchio V, et al. CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons. Curr Biol, 1997,7:112-121.
43 Lu D, Li Y, Wang L, et al. Intraarterial administration of marrow stromal cells in a rat model of traumatic brain injury. J Neurotrauma, 2001,18:813-819.
44 Lu D, Mahmood A, Wang L, et al. Adult bone marrow stromal cells administered intravenously to rats after traumatic brain injury migrate into brain and improve neurological outcome. Neuroreport, 2001,12:559-563.
45 Mahmood A, Lu D, Wang L, et al. Treatment of traumatic brain injury in female rats with intravenous administration of bone marrow stromal cells. Neurosurgery, 2001,49:1196-203; discussion 1203-1204.
46 Chen J, Li Y, Wang L, et al. Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats. J Neurol Sci, 2001,189:49-57.
47 Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke, 2001,32:1005-1011.
48 Wang L, Li Y, Chen J, et al. Ischemic cerebral tissue and MCP-1 enhance rat bone marrow stromal cell migration in interface culture. Exp Hematol, 2002,30:831-836.
49 Askari AT, Unzek S, Popovic ZB, et al. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet, 2003,362:697-703.
50 Kim VN. RNA interference in functional genomics and medicine. J Korean Med Sci, 2003,18:309-318.
51 Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001,411:494-498.
52 Sui G, Soohoo C, Affar el B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci U S A, 2002,99:5515-5520.
53 Shen C, Buck AK, Liu X, et al. Gene silencing by adenovirus-delivered siRNA. FEBS Lett, 2003,539:111-114.
54 Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell, 2002,2:243-247.
55 Tomar RS, Matta H, Chaudhary PM. Use of adeno-associated viral vector for delivery of small interfering RNA. Oncogene, 2003,22:5712-5715.
56 Hommel JD, Sears RM, Georgescu D, et al. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med, 2003,9:1539-1544.
57 Lois C, Refaeli Y, Qin XF, et al. Retroviruses as tools to study the immune system. Curr Opin Immunol, 2001,13:496-504.
58 Wong LF, Goodhead L, Prat C, et al. Lentivirus-mediated gene transfer to the central nervous system: therapeutic and research applications. Hum Gene Ther, 2006,17:1-9.
59 Kordower JH, Emborg ME, Bloch J, et al. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science,2000,290:767-773.
60 Martin-Rendon E, Azzouz M, Mazarakis ND. Lentiviral vectors for the treatment of neurodegenerative diseases. Curr Opin Mol Ther, 2001,3:476-481.
61 Shinagawa T, Ishii S. Generation of Ski-knockdown mice by expressing a long double-strand RNA from an RNA polymerase II promoter. Genes Dev, 2003,17:1340-1345.
62 Tiscornia G, Singer O, Ikawa M, et al. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci U S A, 2003,100:1844-1848.
63 Mittal V. Improving the efficiency of RNA interference in mammals. Nat Rev Genet, 2004,5:355-365.
64 Nishitsuji H, Ikeda T, Miyoshi H, et al. Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary non-dividing cells. Microbes Infect, 2004,6:76-85.
65 Li M, Rossi JJ. Lentiviral vector delivery of siRNA and shRNA encoding genes into cultured and primary hematopoietic cells. Methods Mol Biol, 2005,309:261-272.
66 Wang JS, Shum-Tim D, Galipeau J, et al. Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages. J Thorac Cardiovasc Surg, 2000,120:999-1005.
67 Migliaccio AR, Quarto R, Piacibello W. Cell therapy: filling the gap between basic science and clinical trials October 15-17, 2001, Rome, Italy. Stem Cells, 2003,21:348-356.
68 Lou S, Gu P, Chen F, et al. The effect of bone marrow stromal cells on neuronal differentiation of mesencephalic neural stem cells in Sprague-Dawley rats. Brain Res, 2003,968:114-121.
69 Annabi B, Lee YT, Turcotte S, et al. Hypoxia promotes murine bone-marrow-derived stromal cell migration and tube formation. Stem Cells, 2003,21:337-347.
70 Bittira B, Shum-Tim D, Al-Khaldi A, et al. Mobilization and homing of bone marrow stromal cells in myocardial infarction. Eur J Cardiothorac Surg, 2003,24:393-398.
71 Shichinohe H, Kuroda S, Lee JB, et al. In vivo tracking of bone marrow stromal cells transplanted into mice cerebral infarct by fluorescence optical imaging. Brain Res Brain Res Protoc, 2004,13:166-175.
72 Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow.Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation, 1968,6:230-247.
73 Majumdar MK, Thiede MA, Mosca JD, et al. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells. J Cell Physiol, 1998,176:57-66.
74 Zohar R, Sodek J, McCulloch CA. Characterization of stromal progenitor cells enriched by flow cytometry. Blood, 1997,90:3471-3481.
75 Ratajczak MZ, Kuczynski WI, Sokol DL, et al. Expression and physiologic significance of Kit ligand and stem cell tyrosine kinase-1 receptor ligand in normal human CD34+, c-Kit+ marrow cells. Blood, 1995,86:2161-2167.
76 Li Y, Chen J, Chen XG, et al. Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery. Neurology, 2002,59:514-523.
77 Li Y, Chen J, Wang L, et al. Treatment of stroke in rat with intracarotid administration of marrow stromal cells. Neurology, 2001,56:1666-1672.
78 Miyasaka M, Tanaka T. Lymphocyte trafficking across high endothelial venules: dogmas and enigmas. Nat Rev Immunol, 2004,4:360-370.
79 Wynn RF, Hart CA, Corradi-Perini C, et al. A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. Blood, 2004,104:2643-2645.
80 Von Luttichau I, Notohamiprodjo M, Wechselberger A, et al. Human adult CD34- progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4. Stem Cells Dev, 2005,14:329-336.
81 Sordi V, Malosio ML, Marchesi F, et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood, 2005,106:419-427.
82 Honczarenko M, Le Y, Swierkowski M, et al. Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells, 2006,24:1030-1041.
83 Ruster B, Gottig S, Ludwig RJ, et al. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells. Blood, 2006,108:3938-3944.
84 Chalfie M, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression. Science, 1994,263:802-805.
85 Clayton J. RNA interference: the silent treatment. Nature, 2004,431:599-605.
86 Zhang XN, Xiong W, Wang JD, et al. siRNA-mediated inhibition of HBV replication and expression. World J Gastroenterol, 2004,10:2967-2971.
87 Barton GM, Medzhitov R. Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci U S A, 2002,99:14943-14945.
88 Abbas-Terki T, Blanco-Bose W, Deglon N, et al. Lentiviral-mediated RNA interference. Hum Gene Ther, 2002,13:2197-2201.
89 Paddison PJ, Caudy AA, Hannon GJ. Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci U S A, 2002,99:1443-1448.
90 Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989,20:84-91.
91 Zhang ZG, Bower L, Zhang RL, et al. Three-dimensional measurement of cerebral microvascular plasma perfusion, glial fibrillary acidic protein and microtubule associated protein-2 immunoreactivity after embolic stroke in rats: a double fluorescent labeled laser-scanning confocal microscopic study. Brain Res, 1999,844:55-66.
92 Ortiz LA, Gambelli F, McBride C, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A, 2003,100:8407-8411.
93 Barbash IM, Chouraqui P, Baron J, et al. Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution. Circulation, 2003,108:863-868.
94 Gao J, Dennis JE, Muzic RF, et al. The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs, 2001,169:12-20.
95 Rombouts WJ, Ploemacher RE. Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture. Leukemia, 2003,17:160-170.
96 Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med, 2000,6:1282-1286.
97陈东平,张志坚. SDF-1/CXCR4与骨髓干细胞移植治疗脑卒中.福建医科大学学报, 2007,41:87-89.
98 Chen J, Sanberg PR, Li Y, et al. Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke, 2001,32:2682-2688.
99 Hill WD, Hess DC, Martin-Studdard A, et al. SDF-1 (CXCL12) is upregulated in the ischemic penumbra following stroke: association with bone marrow cell homing to injury. J Neuropathol Exp Neurol JT - Journal of neuropathology and experimental neurology, 2004,63:84-96.
100张志坚,张佳林,陈东平,等.基质细胞衍生因子-1对骨髓间质干细胞迁移的影响.中国临床神经科学, 2006,14:571-576.
101 Ponte AL, Marais E, Gallay N, et al. The in vitro migration capacity of human bonemarrow mesenchymal stem cells: comparison of chemokine and growth factor chemotactic activities. Stem Cells, 2007,25:1737-1745.
102 Aiuti A, Webb IJ, Bleul C, et al. The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J Exp Med, 1997,185:111-120.
103 Kucia M, Ratajczak J, Reca R, et al. Tissue-specific muscle, neural and liver stem/progenitor cells reside in the bone marrow, respond to an SDF-1 gradient and are mobilized into peripheral blood during stress and tissue injury. Blood Cells Mol Dis, 2004,32:52-57.
104 Nanki T, Lipsky PE. Cutting edge: stromal cell-derived factor-1 is a costimulator for CD4+ T cell activation. J Immunol, 2000,164:5010-5014.
105 Abbott JD, Huang Y, Liu D, et al. Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation, 2004,110:3300-3305.
106 Bhakta S, Hong P, Koc O. The surface adhesion molecule CXCR4 stimulates mesenchymal stem cell migration to stromal cell-derived factor-1 in vitro but does not decrease apoptosis under serum deprivation. Cardiovasc Revasc Med, 2006,7:19-24.
107 Stumm RK, Rummel J, Junker V, et al. A dual role for the SDF-1/CXCR4 chemokine receptor system in adult brain: isoform-selective regulation of SDF-1 expression modulates CXCR4-dependent neuronal plasticity and cerebral leukocyte recruitment after focal ischemia. J Neurosci, 2002,22:5865-5878.
108 Sallusto F, Baggiolini M. Chemokines and leukocyte traffic. Nat Immunol, 2008,9:949-952.
109 Fiedler J, Leucht F, Waltenberger J, et al. VEGF-A and PlGF-1 stimulate chemotactic migration of human mesenchymal progenitor cells. Biochem Biophys Res Commun, 2005,334:561-568.
1 Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol, 1976,4:267-274.
2 Horwitz EM, Le Blanc K, Dominici M, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy, 2005,7:393-395.
3 Caplan AI. Mesenchymal stem cells. J Orthop Res, 1991,9:641-650.
4 Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 2002,13:4279-4295.
5 Lee RH, Kim B, Choi I, et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem, 2004,14:311-324.
6 Shih DT, Lee DC, Chen SC, et al. Isolation and characterization of neurogenic mesenchymal stem cells in human scalp tissue. Stem Cells, 2005,23:1012-1020.
7 Toma JG, Akhavan M, Fernandes KJ, et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol, 2001,3:778-784.
8 Fernandez M, Simon V, Herrera G, et al. Detection of stromal cells in peripheral blood progenitor cell collections from breast cancer patients. Bone Marrow Transplant, 1997,20:265-271.
9 In 't Anker PS, Scherjon SA, Kleijburg-van der Keur C, et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells, 2004,22:1338-1345.
10 in 't Anker PS, Noort WA, Scherjon SA, et al. Mesenchymal stem cells in humansecond-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential. Haematologica, 2003,88:845-852.
11 Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Stem Cells, 2004,22:1330-1337.
12 Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol, 2000,109:235-242.
13 Beyer Nardi N, da Silva Meirelles L. Mesenchymal stem cells: isolation, in vitro expansion and characterization. Handb Exp Pharmacol, 2006:249-282.
14 Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999,284:143-147.
15 Sekiya I, Larson BL, Smith JR, et al. Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells, 2002,20:530-541.
16 Gang EJ, Bosnakovski D, Figueiredo CA, et al. SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood, 2007,109:1743-1751.
17 Simmons PJ, Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood, 1991,78:55-62.
18 Haynesworth SE, Baber MA, Caplan AI. Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Bone, 1992,13:69-80.
19 Cheifetz S, Bellon T, Cales C, et al. Endoglin is a component of the transforming growth factor-beta receptor system in human endothelial cells. J Biol Chem, 1992,267:19027-19030.
20 Barry FP, Boynton RE, Haynesworth S, et al. The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105). Biochem Biophys Res Commun, 1999,265:134-139.
21 Bruder SP, Horowitz MC, Mosca JD, et al. Monoclonal antibodies reactive with human osteogenic cell surface antigens. Bone, 1997,21:225-235.
22 Bruder SP, Ricalton NS, Boynton RE, et al. Mesenchymal stem cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation. J Bone Miner Res, 1998,13:655-663.
23 Majumdar MK, Keane-Moore M, Buyaner D, et al. Characterization and functionality of cell surface molecules on human mesenchymal stem cells. J Biomed Sci, 2003,10:228-241.
24 Honczarenko M, Le Y, Swierkowski M, et al. Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells,2006,24:1030-1041.
25 Jaiswal RK, Jaiswal N, Bruder SP, et al. Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem, 2000,275:9645-9652.
26 Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci U S A, 1999,96:10711-10716.
27 Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest, 1999,103:697-705.
28 Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science, 1998,279:1528-1530.
29 Yang Y, Relan NK, Przywara DA, et al. Embryonic mesenchymal cells share the potential for smooth muscle differentiation: myogenesis is controlled by the cell's shape. Development, 1999,126:3027-3033.
30 Minguell JJ, Erices A, Conget P. Mesenchymal stem cells. Exp Biol Med (Maywood), 2001,226:507-520.
31 Tondreau T, Lagneaux L, Dejeneffe M, et al. Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation. Differentiation, 2004,72:319-326.
32 Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med, 1999,5:309-313.
33 Horwitz EM, Prockop DJ, Gordon PL, et al. Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood, 2001,97:1227-31.
34 Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet, 2004,363:1439-1441.
35 Chen SL, Fang WW, Qian J, et al. Improvement of cardiac function after transplantation of autologous bone marrow mesenchymal stem cells in patients with acute myocardial infarction. Chin Med J (Engl), 2004,117:1443-1448.
36 Ramasamy R, Lam EW, Soeiro I, et al. Mesenchymal stem cells inhibit proliferation and apoptosis of tumor cells: impact on in vivo tumor growth. Leukemia, 2007,21:304-310.
37 Barbash IM, Chouraqui P, Baron J, et al. Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution. Circulation, 2003,108:863-868.
38 McFarlin K, Gao X, Liu YB, et al. Bone marrow-derived mesenchymal stromal cellsaccelerate wound healing in the rat. Wound Repair Regen, 2006,14:471-478.
39 Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke, 2001,32:1005-1011.
40 Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proc Natl Acad Sci U S A, 2002,99:8932-8937.
41 Ortiz LA, Gambelli F, McBride C, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A, 2003,100:8407-8411.
42 Wu GD, Nolta JA, Jin YS, et al. Migration of mesenchymal stem cells to heart allografts during chronic rejection. Transplantation, 2003,75:679-685.
43 Rombouts WJ, Ploemacher RE. Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture. Leukemia, 2003,17:160-170.
44 Gao J, Dennis JE, Muzic RF, et al. The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs, 2001,169:12-20.
45 Yu S, Tanabe T, Dezawa M, et al. Effects of bone marrow stromal cell injection in an experimental glaucoma model. Biochem Biophys Res Commun, 2006,344:1071-1079.
46 Yamada Y, Ueda M, Hibi H, et al. A novel approach to periodontal tissue regeneration with mesenchymal stem cells and platelet-rich plasma using tissue engineering technology: A clinical case report. Int J Periodontics Restorative Dent, 2006,26:363-369.
47 Miyahara Y, Nagaya N, Kataoka M, et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med, 2006,12:459-465.
48 Hou M, Yang KM, Zhang H, et al. Transplantation of mesenchymal stem cells from human bone marrow improves damaged heart function in rats. Int J Cardiol, 2007,115:220-228.
49 Pereira RF, Halford KW, O'Hara MD, et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci U S A, 1995,92:4857-4861.
50 Elzaouk L, Moelling K, Pavlovic J. Anti-tumor activity of mesenchymal stem cells producing IL-12 in a mouse melanoma model. Exp Dermatol, 2006,15:865-874.
51 Bulte JW, Douglas T, Witwer B, et al. Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells. Nat Biotechnol, 2001,19:1141-1147.
52 Arbab AS, Jordan EK, Wilson LB, et al. In vivo trafficking and targeted delivery of magnetically labeled stem cells. Hum Gene Ther, 2004,15:351-360.
53 Kraitchman DL, Heldman AW, Atalar E, et al. In vivo magnetic resonance imaging ofmesenchymal stem cells in myocardial infarction. Circulation, 2003,107:2290-2293.
54 Arbab AS, Yocum GT, Kalish H, et al. Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI. Blood, 2004,104:1217-1223.
55 Arbab AS, Yocum GT, Rad AM, et al. Labeling of cells with ferumoxides-protamine sulfate complexes does not inhibit function or differentiation capacity of hematopoietic or mesenchymal stem cells. NMR Biomed, 2005,18:553-559.
56 Bulte JW, Kraitchman DL, Mackay AM, et al. Chondrogenic differentiation of mesenchymal stem cells is inhibited after magnetic labeling with ferumoxides. Blood, 2004,104:3410-2; author reply 3412-3413.
57 Bos C, Delmas Y, Desmouliere A, et al. In vivo MR imaging of intravascularly injected magnetically labeled mesenchymal stem cells in rat kidney and liver. Radiology, 2004,233:781-789.
58 Hauger O, Frost EE, van Heeswijk R, et al. MR evaluation of the glomerular homing of magnetically labeled mesenchymal stem cells in a rat model of nephropathy. Radiology, 2006,238:200-210.
59 Dai W, Hale SL, Martin BJ, et al. Allogeneic mesenchymal stem cell transplantation in postinfarcted rat myocardium: short- and long-term effects. Circulation, 2005,112:214-23.
60 Li GR, Deng XL, Sun H, et al. Ion channels in mesenchymal stem cells from rat bone marrow. Stem Cells, 2006,24:1519-1528.
61 Pijnappels DA, Schalij MJ, van Tuyn J, et al. Progressive increase in conduction velocity across human mesenchymal stem cells is mediated by enhanced electrical coupling. Cardiovasc Res, 2006,72:282-291.
62 Choi CB, Cho YK, Prakash KV, et al. Analysis of neuron-like differentiation of human bone marrow mesenchymal stem cells. Biochem Biophys Res Commun, 2006,350:138-146.
63 Noiseux N, Gnecchi M, Lopez-Ilasaca M, et al. Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiation. Mol Ther, 2006,14:840-850.
64 Krampera M, Marconi S, Pasini A, et al. Induction of neural-like differentiation in human mesenchymal stem cells derived from bone marrow, fat, spleen and thymus. Bone, 2007,40:382-390.
65 Togel F, Hu Z, Weiss K, et al. Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am J Physiol Renal Physiol, 2005,289:F31-42.
66 Rasmusson I. Immune modulation by mesenchymal stem cells. Exp Cell Res, 2006,312:2169-2179.
67 Gnecchi M, He H, Liang OD, et al. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med, 2005,11:367-368.
68 Gnecchi M, He H, Noiseux N, et al. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J, 2006,20:661-669.
69 Weimar IS, Miranda N, Muller EJ, et al. Hepatocyte growth factor/scatter factor (HGF/SF) is produced by human bone marrow stromal cells and promotes proliferation, adhesion and survival of human hematopoietic progenitor cells (CD34+). Exp Hematol, 1998,26:885-894.
70 Ivanova N, Dobrin R, Lu R, et al. Dissecting self-renewal in stem cells with RNA interference. Nature, 2006,442:533-538.
71 Xu Y, Mirmalek-Sani SH, Yang X, et al. The use of small interfering RNAs to inhibit adipocyte differentiation in human preadipocytes and fetal-femur-derived mesenchymal cells. Exp Cell Res, 2006,312:1856-1864.
72 Yoon J, Min BG, Kim YH, et al. Differentiation, engraftment and functional effects of pre-treated mesenchymal stem cells in a rat myocardial infarct model. Acta Cardiol, 2005,60:277-284.
73 Butcher EC. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell, 1991,67:1033-1036.
74 Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell, 1994,76:301-314.
75 Chute JP. Stem cell homing. Curr Opin Hematol, 2006,13:399-406.
76 Ruster B, Gottig S, Ludwig RJ, et al. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells. Blood, 2006,108:3938-3944.
77 Muller WA, Weigl SA, Deng X, et al. PECAM-1 is required for transendothelial migration of leukocytes. J Exp Med, 1993,178:449-460.
78 Bruder SP, Jaiswal N, Ricalton NS, et al. Mesenchymal stem cells in osteobiology and applied bone regeneration. Clin Orthop Relat Res, 1998:S247-256.
79 Miyasaka M, Tanaka T. Lymphocyte trafficking across high endothelial venules: dogmas and enigmas. Nat Rev Immunol, 2004,4:360-370.
80 Wynn RF, Hart CA, Corradi-Perini C, et al. A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. Blood, 2004,104:2643-2645.
81 Von Luttichau I, Notohamiprodjo M, Wechselberger A, et al. Human adult CD34- progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4. Stem Cells Dev, 2005,14:329-336.
82 Sordi V, Malosio ML, Marchesi F, et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood, 2005,106:419-427.
83 Ji JF, He BP, Dheen ST, et al. Interactions of chemokines and chemokine receptors mediate the migration of mesenchymal stem cells to the impaired site in the brain after hypoglossal nerve injury. Stem Cells, 2004,22:415-427.
84 Kortesidis A, Zannettino A, Isenmann S, et al. Stromal-derived factor-1 promotes the growth, survival, and development of human bone marrow stromal stem cells. Blood, 2005,105:3793-3801.
85 Abbott JD, Huang Y, Liu D, et al. Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation, 2004,110:3300-3305.
86 Wang Y, Johnsen HE, Mortensen S, et al. Changes in circulating mesenchymal stem cells, stem cell homing factor, and vascular growth factors in patients with acute ST elevation myocardial infarction treated with primary percutaneous coronary intervention. Heart, 2006,92:768-774.
87 Wang L, Li Y, Chen J, et al. Ischemic cerebral tissue and MCP-1 enhance rat bone marrow stromal cell migration in interface culture. Exp Hematol, 2002,30:831-836.
88 Wang L, Li Y, Chen X, et al. MCP-1, MIP-1, IL-8 and ischemic cerebral tissue enhance human bone marrow stromal cell migration in interface culture. Hematology, 2002,7:113-117.
89 Batten P, Sarathchandra P, Antoniw JW, et al. Human mesenchymal stem cells induce T cell anergy and downregulate T cell allo-responses via the TH2 pathway: relevance to tissue engineering human heart valves. Tissue Eng, 2006,12:2263-2273.
90 Segers VF, Van Riet I, Andries LJ, et al. Mesenchymal stem cell adhesion to cardiac microvascular endothelium: activators and mechanisms. Am J Physiol Heart Circ Physiol, 2006,290:H1370-1377.
91 Schmidt A, Ladage D, Steingen C, et al. Mesenchymal stem cells transmigrate over the endothelial barrier. Eur J Cell Biol, 2006,85:1179-1188.
92 Devine SM, Bartholomew AM, Mahmud N, et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol, 2001,29:244-255.
93 Jiang WH, Ma AQ, Han K, et al. [Ultrastructural changes of rat ischemic myocardium after bone marrow mesenchymal stem cell transplantation]. Di Yi Jun Yi Da Xue Xue Bao, 2005,25:1090-1094.
2 Fox JM, Chamberlain G, Ashton BA, et al. Recent advances into the understanding of mesenchymal stem cell trafficking. Br J Haematol, 2007,137:491-502.
3 Brooke G, Cook M, Blair C, et al. Therapeutic applications of mesenchymal stromal cells. Semin Cell Dev Biol, 2007,18:846-858.
4 Shyu WC, Lee YJ, Liu DD, et al. Homing genes, cell therapy and stroke. Front Biosci, 2006,11:899-907.
5 Chen J, Li Y, Wang L, et al. Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats. J Neurol Sci JT - Journal of the neurological sciences, 2001,189:49-57.
6 Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke JT - Stroke; a journal of cerebral circulation, 2001,32:1005-1011.
7 Lu D, Mahmood A, Wang L, et al. Adult bone marrow stromal cells administered intravenously to rats after traumatic brain injury migrate into brain and improve neurological outcome. Neuroreport JT - Neuroreport, 2001,12:559-563.
8 Lee RH, Hsu SC, Munoz J, et al. A subset of human rapidly self-renewing marrow stromal cells preferentially engraft in mice. Blood, 2006,107:2153-2161.
9 Kim CH, Broxmeyer HE. Chemokines: signal lamps for trafficking of T and B cells for development and effector function. J Leukoc Biol, 1999,65:6-15.
10 Hattori K, Heissig B, Rafii S. The regulation of hematopoietic stem cell and progenitor mobilization by chemokine SDF-1. Leuk Lymphoma JT - Leukemia & lymphoma, 2003,44:575-582.
11 Salvucci O, Yao L, Villalba S, et al. Regulation of endothelial cell branching morphogenesis by endogenous chemokine stromal-derived factor-1. Blood JT - Blood, 2002,99:2703-2711.
12 Yamaguchi J, Kusano KF, Masuo O, et al. Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization. Circulation JT - Circulation, 2003,107:1322-1328.
13 Tham TN, Lazarini F, Franceschini IA, et al. Developmental pattern of expression of the alpha chemokine stromal cell-derived factor 1 in the rat central nervous system. Eur J Neurosci JT - The European journal of neuroscience, 2001,13:845-856.
14 Petit I, Szyper-Kravitz M, Nagler A, et al. G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol JT - Nature immunology, 2002,3:687-694.
15 Stumm RK, Rummel J, Junker V, et al. A dual role for the SDF-1/CXCR4 chemokine receptor system in adult brain: isoform-selective regulation of SDF-1 expression modulates CXCR4-dependent neuronal plasticity and cerebral leukocyte recruitment after focal ischemia. J Neurosci JT - The Journal of neuroscience : the official journal of the Society for Neuroscience, 2002,22:5865-5878.
16 Askari AT, Unzek S, Popovic ZB, et al. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet JT - Lancet, 2003,362:697-703.
17 Downward J. RNA interference. BMJ, 2004,328:1245-1248.
18 Scherr M, Morgan MA, Eder M. Gene silencing mediated by small interfering RNAs in mammalian cells. Curr Med Chem, 2003,10:245-256.
19 Rubinson DA, Dillon CP, Kwiatkowski AV, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet, 2003,33:401-406.
20 Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA, 2003,9:493-501.
21 Baggiolini M. Chemokines and leukocyte traffic. Nature, 1998,392:565-568.
22 Melchers F, Rolink AG, Schaniel C. The role of chemokines in regulating cell migration during humoral immune responses. Cell, 1999,99:351-354.
23 Murdoch C, Finn A. Chemokine receptors and their role in inflammation and infectious diseases. Blood, 2000,95:3032-3043.
24 Bazan JF, Bacon KB, Hardiman G, et al. A new class of membrane-bound chemokine with a CX3C motif. Nature, 1997,385:640-644.
25 Kelner GS, Kennedy J, Bacon KB, et al. Lymphotactin: a cytokine that represents a new class of chemokine. Science, 1994,266:1395-1399.
26 Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity, 2000,12:121-127.
27 Wang L, Li Y, Chen X, et al. MCP-1, MIP-1, IL-8 and ischemic cerebral tissue enhance human bone marrow stromal cell migration in interface culture. Hematology, 2002,7:113-117.
28 Ji JF, He BP, Dheen ST, et al. Interactions of chemokines and chemokine receptors mediate the migration of mesenchymal stem cells to the impaired site in the brain afterhypoglossal nerve injury. Stem Cells, 2004,22:415-427.
29 Wang Y, Deng Y, Zhou GQ. SDF-1alpha/CXCR4-mediated migration of systemically transplanted bone marrow stromal cells towards ischemic brain lesion in a rat model. Brain Res, 2008,1195:104-112.
30 Hill WD, Hess DC, Martin-Studdard A, et al. SDF-1 (CXCL12) is upregulated in the ischemic penumbra following stroke: association with bone marrow cell homing to injury. J Neuropathol Exp Neurol, 2004,63:84-96.
31 Cui X, Chen J, Zacharek A, et al. Nitric oxide donor upregulation of stromal cell-derived factor-1/chemokine (CXC motif) receptor 4 enhances bone marrow stromal cell migration into ischemic brain after stroke. Stem Cells, 2007,25:2777-2785.
32 Shirozu M, Nakano T, Inazawa J, et al. Structure and chromosomal localization of the human stromal cell-derived factor 1 (SDF1) gene. Genomics, 1995,28:495-500.
33 Loetscher M, Geiser T, O'Reilly T, et al. Cloning of a human seven-transmembrane domain receptor, LESTR, that is highly expressed in leukocytes. J Biol Chem, 1994,269:232-237.
34 Feng Y, Broder CC, Kennedy PE, et al. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science, 1996,272:872-877.
35 Horuk R. Chemokines beyond inflammation. Nature, 1998,393:524-525.
36 Horuk R. Chemokine receptors. Cytokine Growth Factor Rev, 2001,12:313-335.
37 Muller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature, 2001,410:50-56.
38 Scotton CJ, Wilson JL, Scott K, et al. Multiple actions of the chemokine CXCL12 on epithelial tumor cells in human ovarian cancer. Cancer Res, 2002,62:5930-5938.
39 Hideshima T, Chauhan D, Hayashi T, et al. The biological sequelae of stromal cell-derived factor-1alpha in multiple myeloma. Mol Cancer Ther, 2002,1:539-544.
40 Taichman RS, Cooper C, Keller ET, et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone. Cancer Res, 2002,62:1832-1837.
41 Peled A, Petit I, Kollet O, et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science, 1999,283:845-848.
42 Hesselgesser J, Halks-Miller M, DelVecchio V, et al. CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons. Curr Biol, 1997,7:112-121.
43 Lu D, Li Y, Wang L, et al. Intraarterial administration of marrow stromal cells in a rat model of traumatic brain injury. J Neurotrauma, 2001,18:813-819.
44 Lu D, Mahmood A, Wang L, et al. Adult bone marrow stromal cells administered intravenously to rats after traumatic brain injury migrate into brain and improve neurological outcome. Neuroreport, 2001,12:559-563.
45 Mahmood A, Lu D, Wang L, et al. Treatment of traumatic brain injury in female rats with intravenous administration of bone marrow stromal cells. Neurosurgery, 2001,49:1196-203; discussion 1203-1204.
46 Chen J, Li Y, Wang L, et al. Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats. J Neurol Sci, 2001,189:49-57.
47 Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke, 2001,32:1005-1011.
48 Wang L, Li Y, Chen J, et al. Ischemic cerebral tissue and MCP-1 enhance rat bone marrow stromal cell migration in interface culture. Exp Hematol, 2002,30:831-836.
49 Askari AT, Unzek S, Popovic ZB, et al. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet, 2003,362:697-703.
50 Kim VN. RNA interference in functional genomics and medicine. J Korean Med Sci, 2003,18:309-318.
51 Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001,411:494-498.
52 Sui G, Soohoo C, Affar el B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci U S A, 2002,99:5515-5520.
53 Shen C, Buck AK, Liu X, et al. Gene silencing by adenovirus-delivered siRNA. FEBS Lett, 2003,539:111-114.
54 Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell, 2002,2:243-247.
55 Tomar RS, Matta H, Chaudhary PM. Use of adeno-associated viral vector for delivery of small interfering RNA. Oncogene, 2003,22:5712-5715.
56 Hommel JD, Sears RM, Georgescu D, et al. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med, 2003,9:1539-1544.
57 Lois C, Refaeli Y, Qin XF, et al. Retroviruses as tools to study the immune system. Curr Opin Immunol, 2001,13:496-504.
58 Wong LF, Goodhead L, Prat C, et al. Lentivirus-mediated gene transfer to the central nervous system: therapeutic and research applications. Hum Gene Ther, 2006,17:1-9.
59 Kordower JH, Emborg ME, Bloch J, et al. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science,2000,290:767-773.
60 Martin-Rendon E, Azzouz M, Mazarakis ND. Lentiviral vectors for the treatment of neurodegenerative diseases. Curr Opin Mol Ther, 2001,3:476-481.
61 Shinagawa T, Ishii S. Generation of Ski-knockdown mice by expressing a long double-strand RNA from an RNA polymerase II promoter. Genes Dev, 2003,17:1340-1345.
62 Tiscornia G, Singer O, Ikawa M, et al. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci U S A, 2003,100:1844-1848.
63 Mittal V. Improving the efficiency of RNA interference in mammals. Nat Rev Genet, 2004,5:355-365.
64 Nishitsuji H, Ikeda T, Miyoshi H, et al. Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary non-dividing cells. Microbes Infect, 2004,6:76-85.
65 Li M, Rossi JJ. Lentiviral vector delivery of siRNA and shRNA encoding genes into cultured and primary hematopoietic cells. Methods Mol Biol, 2005,309:261-272.
66 Wang JS, Shum-Tim D, Galipeau J, et al. Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages. J Thorac Cardiovasc Surg, 2000,120:999-1005.
67 Migliaccio AR, Quarto R, Piacibello W. Cell therapy: filling the gap between basic science and clinical trials October 15-17, 2001, Rome, Italy. Stem Cells, 2003,21:348-356.
68 Lou S, Gu P, Chen F, et al. The effect of bone marrow stromal cells on neuronal differentiation of mesencephalic neural stem cells in Sprague-Dawley rats. Brain Res, 2003,968:114-121.
69 Annabi B, Lee YT, Turcotte S, et al. Hypoxia promotes murine bone-marrow-derived stromal cell migration and tube formation. Stem Cells, 2003,21:337-347.
70 Bittira B, Shum-Tim D, Al-Khaldi A, et al. Mobilization and homing of bone marrow stromal cells in myocardial infarction. Eur J Cardiothorac Surg, 2003,24:393-398.
71 Shichinohe H, Kuroda S, Lee JB, et al. In vivo tracking of bone marrow stromal cells transplanted into mice cerebral infarct by fluorescence optical imaging. Brain Res Brain Res Protoc, 2004,13:166-175.
72 Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow.Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation, 1968,6:230-247.
73 Majumdar MK, Thiede MA, Mosca JD, et al. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells. J Cell Physiol, 1998,176:57-66.
74 Zohar R, Sodek J, McCulloch CA. Characterization of stromal progenitor cells enriched by flow cytometry. Blood, 1997,90:3471-3481.
75 Ratajczak MZ, Kuczynski WI, Sokol DL, et al. Expression and physiologic significance of Kit ligand and stem cell tyrosine kinase-1 receptor ligand in normal human CD34+, c-Kit+ marrow cells. Blood, 1995,86:2161-2167.
76 Li Y, Chen J, Chen XG, et al. Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery. Neurology, 2002,59:514-523.
77 Li Y, Chen J, Wang L, et al. Treatment of stroke in rat with intracarotid administration of marrow stromal cells. Neurology, 2001,56:1666-1672.
78 Miyasaka M, Tanaka T. Lymphocyte trafficking across high endothelial venules: dogmas and enigmas. Nat Rev Immunol, 2004,4:360-370.
79 Wynn RF, Hart CA, Corradi-Perini C, et al. A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. Blood, 2004,104:2643-2645.
80 Von Luttichau I, Notohamiprodjo M, Wechselberger A, et al. Human adult CD34- progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4. Stem Cells Dev, 2005,14:329-336.
81 Sordi V, Malosio ML, Marchesi F, et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood, 2005,106:419-427.
82 Honczarenko M, Le Y, Swierkowski M, et al. Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells, 2006,24:1030-1041.
83 Ruster B, Gottig S, Ludwig RJ, et al. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells. Blood, 2006,108:3938-3944.
84 Chalfie M, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression. Science, 1994,263:802-805.
85 Clayton J. RNA interference: the silent treatment. Nature, 2004,431:599-605.
86 Zhang XN, Xiong W, Wang JD, et al. siRNA-mediated inhibition of HBV replication and expression. World J Gastroenterol, 2004,10:2967-2971.
87 Barton GM, Medzhitov R. Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci U S A, 2002,99:14943-14945.
88 Abbas-Terki T, Blanco-Bose W, Deglon N, et al. Lentiviral-mediated RNA interference. Hum Gene Ther, 2002,13:2197-2201.
89 Paddison PJ, Caudy AA, Hannon GJ. Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci U S A, 2002,99:1443-1448.
90 Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989,20:84-91.
91 Zhang ZG, Bower L, Zhang RL, et al. Three-dimensional measurement of cerebral microvascular plasma perfusion, glial fibrillary acidic protein and microtubule associated protein-2 immunoreactivity after embolic stroke in rats: a double fluorescent labeled laser-scanning confocal microscopic study. Brain Res, 1999,844:55-66.
92 Ortiz LA, Gambelli F, McBride C, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A, 2003,100:8407-8411.
93 Barbash IM, Chouraqui P, Baron J, et al. Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution. Circulation, 2003,108:863-868.
94 Gao J, Dennis JE, Muzic RF, et al. The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs, 2001,169:12-20.
95 Rombouts WJ, Ploemacher RE. Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture. Leukemia, 2003,17:160-170.
96 Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med, 2000,6:1282-1286.
97陈东平,张志坚. SDF-1/CXCR4与骨髓干细胞移植治疗脑卒中.福建医科大学学报, 2007,41:87-89.
98 Chen J, Sanberg PR, Li Y, et al. Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke, 2001,32:2682-2688.
99 Hill WD, Hess DC, Martin-Studdard A, et al. SDF-1 (CXCL12) is upregulated in the ischemic penumbra following stroke: association with bone marrow cell homing to injury. J Neuropathol Exp Neurol JT - Journal of neuropathology and experimental neurology, 2004,63:84-96.
100张志坚,张佳林,陈东平,等.基质细胞衍生因子-1对骨髓间质干细胞迁移的影响.中国临床神经科学, 2006,14:571-576.
101 Ponte AL, Marais E, Gallay N, et al. The in vitro migration capacity of human bonemarrow mesenchymal stem cells: comparison of chemokine and growth factor chemotactic activities. Stem Cells, 2007,25:1737-1745.
102 Aiuti A, Webb IJ, Bleul C, et al. The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J Exp Med, 1997,185:111-120.
103 Kucia M, Ratajczak J, Reca R, et al. Tissue-specific muscle, neural and liver stem/progenitor cells reside in the bone marrow, respond to an SDF-1 gradient and are mobilized into peripheral blood during stress and tissue injury. Blood Cells Mol Dis, 2004,32:52-57.
104 Nanki T, Lipsky PE. Cutting edge: stromal cell-derived factor-1 is a costimulator for CD4+ T cell activation. J Immunol, 2000,164:5010-5014.
105 Abbott JD, Huang Y, Liu D, et al. Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation, 2004,110:3300-3305.
106 Bhakta S, Hong P, Koc O. The surface adhesion molecule CXCR4 stimulates mesenchymal stem cell migration to stromal cell-derived factor-1 in vitro but does not decrease apoptosis under serum deprivation. Cardiovasc Revasc Med, 2006,7:19-24.
107 Stumm RK, Rummel J, Junker V, et al. A dual role for the SDF-1/CXCR4 chemokine receptor system in adult brain: isoform-selective regulation of SDF-1 expression modulates CXCR4-dependent neuronal plasticity and cerebral leukocyte recruitment after focal ischemia. J Neurosci, 2002,22:5865-5878.
108 Sallusto F, Baggiolini M. Chemokines and leukocyte traffic. Nat Immunol, 2008,9:949-952.
109 Fiedler J, Leucht F, Waltenberger J, et al. VEGF-A and PlGF-1 stimulate chemotactic migration of human mesenchymal progenitor cells. Biochem Biophys Res Commun, 2005,334:561-568.
1 Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol, 1976,4:267-274.
2 Horwitz EM, Le Blanc K, Dominici M, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy, 2005,7:393-395.
3 Caplan AI. Mesenchymal stem cells. J Orthop Res, 1991,9:641-650.
4 Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 2002,13:4279-4295.
5 Lee RH, Kim B, Choi I, et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem, 2004,14:311-324.
6 Shih DT, Lee DC, Chen SC, et al. Isolation and characterization of neurogenic mesenchymal stem cells in human scalp tissue. Stem Cells, 2005,23:1012-1020.
7 Toma JG, Akhavan M, Fernandes KJ, et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol, 2001,3:778-784.
8 Fernandez M, Simon V, Herrera G, et al. Detection of stromal cells in peripheral blood progenitor cell collections from breast cancer patients. Bone Marrow Transplant, 1997,20:265-271.
9 In 't Anker PS, Scherjon SA, Kleijburg-van der Keur C, et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells, 2004,22:1338-1345.
10 in 't Anker PS, Noort WA, Scherjon SA, et al. Mesenchymal stem cells in humansecond-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential. Haematologica, 2003,88:845-852.
11 Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Stem Cells, 2004,22:1330-1337.
12 Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol, 2000,109:235-242.
13 Beyer Nardi N, da Silva Meirelles L. Mesenchymal stem cells: isolation, in vitro expansion and characterization. Handb Exp Pharmacol, 2006:249-282.
14 Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999,284:143-147.
15 Sekiya I, Larson BL, Smith JR, et al. Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells, 2002,20:530-541.
16 Gang EJ, Bosnakovski D, Figueiredo CA, et al. SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood, 2007,109:1743-1751.
17 Simmons PJ, Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood, 1991,78:55-62.
18 Haynesworth SE, Baber MA, Caplan AI. Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Bone, 1992,13:69-80.
19 Cheifetz S, Bellon T, Cales C, et al. Endoglin is a component of the transforming growth factor-beta receptor system in human endothelial cells. J Biol Chem, 1992,267:19027-19030.
20 Barry FP, Boynton RE, Haynesworth S, et al. The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105). Biochem Biophys Res Commun, 1999,265:134-139.
21 Bruder SP, Horowitz MC, Mosca JD, et al. Monoclonal antibodies reactive with human osteogenic cell surface antigens. Bone, 1997,21:225-235.
22 Bruder SP, Ricalton NS, Boynton RE, et al. Mesenchymal stem cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation. J Bone Miner Res, 1998,13:655-663.
23 Majumdar MK, Keane-Moore M, Buyaner D, et al. Characterization and functionality of cell surface molecules on human mesenchymal stem cells. J Biomed Sci, 2003,10:228-241.
24 Honczarenko M, Le Y, Swierkowski M, et al. Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells,2006,24:1030-1041.
25 Jaiswal RK, Jaiswal N, Bruder SP, et al. Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem, 2000,275:9645-9652.
26 Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci U S A, 1999,96:10711-10716.
27 Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest, 1999,103:697-705.
28 Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science, 1998,279:1528-1530.
29 Yang Y, Relan NK, Przywara DA, et al. Embryonic mesenchymal cells share the potential for smooth muscle differentiation: myogenesis is controlled by the cell's shape. Development, 1999,126:3027-3033.
30 Minguell JJ, Erices A, Conget P. Mesenchymal stem cells. Exp Biol Med (Maywood), 2001,226:507-520.
31 Tondreau T, Lagneaux L, Dejeneffe M, et al. Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation. Differentiation, 2004,72:319-326.
32 Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med, 1999,5:309-313.
33 Horwitz EM, Prockop DJ, Gordon PL, et al. Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood, 2001,97:1227-31.
34 Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet, 2004,363:1439-1441.
35 Chen SL, Fang WW, Qian J, et al. Improvement of cardiac function after transplantation of autologous bone marrow mesenchymal stem cells in patients with acute myocardial infarction. Chin Med J (Engl), 2004,117:1443-1448.
36 Ramasamy R, Lam EW, Soeiro I, et al. Mesenchymal stem cells inhibit proliferation and apoptosis of tumor cells: impact on in vivo tumor growth. Leukemia, 2007,21:304-310.
37 Barbash IM, Chouraqui P, Baron J, et al. Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution. Circulation, 2003,108:863-868.
38 McFarlin K, Gao X, Liu YB, et al. Bone marrow-derived mesenchymal stromal cellsaccelerate wound healing in the rat. Wound Repair Regen, 2006,14:471-478.
39 Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke, 2001,32:1005-1011.
40 Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proc Natl Acad Sci U S A, 2002,99:8932-8937.
41 Ortiz LA, Gambelli F, McBride C, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A, 2003,100:8407-8411.
42 Wu GD, Nolta JA, Jin YS, et al. Migration of mesenchymal stem cells to heart allografts during chronic rejection. Transplantation, 2003,75:679-685.
43 Rombouts WJ, Ploemacher RE. Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture. Leukemia, 2003,17:160-170.
44 Gao J, Dennis JE, Muzic RF, et al. The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs, 2001,169:12-20.
45 Yu S, Tanabe T, Dezawa M, et al. Effects of bone marrow stromal cell injection in an experimental glaucoma model. Biochem Biophys Res Commun, 2006,344:1071-1079.
46 Yamada Y, Ueda M, Hibi H, et al. A novel approach to periodontal tissue regeneration with mesenchymal stem cells and platelet-rich plasma using tissue engineering technology: A clinical case report. Int J Periodontics Restorative Dent, 2006,26:363-369.
47 Miyahara Y, Nagaya N, Kataoka M, et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med, 2006,12:459-465.
48 Hou M, Yang KM, Zhang H, et al. Transplantation of mesenchymal stem cells from human bone marrow improves damaged heart function in rats. Int J Cardiol, 2007,115:220-228.
49 Pereira RF, Halford KW, O'Hara MD, et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci U S A, 1995,92:4857-4861.
50 Elzaouk L, Moelling K, Pavlovic J. Anti-tumor activity of mesenchymal stem cells producing IL-12 in a mouse melanoma model. Exp Dermatol, 2006,15:865-874.
51 Bulte JW, Douglas T, Witwer B, et al. Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells. Nat Biotechnol, 2001,19:1141-1147.
52 Arbab AS, Jordan EK, Wilson LB, et al. In vivo trafficking and targeted delivery of magnetically labeled stem cells. Hum Gene Ther, 2004,15:351-360.
53 Kraitchman DL, Heldman AW, Atalar E, et al. In vivo magnetic resonance imaging ofmesenchymal stem cells in myocardial infarction. Circulation, 2003,107:2290-2293.
54 Arbab AS, Yocum GT, Kalish H, et al. Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI. Blood, 2004,104:1217-1223.
55 Arbab AS, Yocum GT, Rad AM, et al. Labeling of cells with ferumoxides-protamine sulfate complexes does not inhibit function or differentiation capacity of hematopoietic or mesenchymal stem cells. NMR Biomed, 2005,18:553-559.
56 Bulte JW, Kraitchman DL, Mackay AM, et al. Chondrogenic differentiation of mesenchymal stem cells is inhibited after magnetic labeling with ferumoxides. Blood, 2004,104:3410-2; author reply 3412-3413.
57 Bos C, Delmas Y, Desmouliere A, et al. In vivo MR imaging of intravascularly injected magnetically labeled mesenchymal stem cells in rat kidney and liver. Radiology, 2004,233:781-789.
58 Hauger O, Frost EE, van Heeswijk R, et al. MR evaluation of the glomerular homing of magnetically labeled mesenchymal stem cells in a rat model of nephropathy. Radiology, 2006,238:200-210.
59 Dai W, Hale SL, Martin BJ, et al. Allogeneic mesenchymal stem cell transplantation in postinfarcted rat myocardium: short- and long-term effects. Circulation, 2005,112:214-23.
60 Li GR, Deng XL, Sun H, et al. Ion channels in mesenchymal stem cells from rat bone marrow. Stem Cells, 2006,24:1519-1528.
61 Pijnappels DA, Schalij MJ, van Tuyn J, et al. Progressive increase in conduction velocity across human mesenchymal stem cells is mediated by enhanced electrical coupling. Cardiovasc Res, 2006,72:282-291.
62 Choi CB, Cho YK, Prakash KV, et al. Analysis of neuron-like differentiation of human bone marrow mesenchymal stem cells. Biochem Biophys Res Commun, 2006,350:138-146.
63 Noiseux N, Gnecchi M, Lopez-Ilasaca M, et al. Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiation. Mol Ther, 2006,14:840-850.
64 Krampera M, Marconi S, Pasini A, et al. Induction of neural-like differentiation in human mesenchymal stem cells derived from bone marrow, fat, spleen and thymus. Bone, 2007,40:382-390.
65 Togel F, Hu Z, Weiss K, et al. Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am J Physiol Renal Physiol, 2005,289:F31-42.
66 Rasmusson I. Immune modulation by mesenchymal stem cells. Exp Cell Res, 2006,312:2169-2179.
67 Gnecchi M, He H, Liang OD, et al. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med, 2005,11:367-368.
68 Gnecchi M, He H, Noiseux N, et al. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J, 2006,20:661-669.
69 Weimar IS, Miranda N, Muller EJ, et al. Hepatocyte growth factor/scatter factor (HGF/SF) is produced by human bone marrow stromal cells and promotes proliferation, adhesion and survival of human hematopoietic progenitor cells (CD34+). Exp Hematol, 1998,26:885-894.
70 Ivanova N, Dobrin R, Lu R, et al. Dissecting self-renewal in stem cells with RNA interference. Nature, 2006,442:533-538.
71 Xu Y, Mirmalek-Sani SH, Yang X, et al. The use of small interfering RNAs to inhibit adipocyte differentiation in human preadipocytes and fetal-femur-derived mesenchymal cells. Exp Cell Res, 2006,312:1856-1864.
72 Yoon J, Min BG, Kim YH, et al. Differentiation, engraftment and functional effects of pre-treated mesenchymal stem cells in a rat myocardial infarct model. Acta Cardiol, 2005,60:277-284.
73 Butcher EC. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell, 1991,67:1033-1036.
74 Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell, 1994,76:301-314.
75 Chute JP. Stem cell homing. Curr Opin Hematol, 2006,13:399-406.
76 Ruster B, Gottig S, Ludwig RJ, et al. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells. Blood, 2006,108:3938-3944.
77 Muller WA, Weigl SA, Deng X, et al. PECAM-1 is required for transendothelial migration of leukocytes. J Exp Med, 1993,178:449-460.
78 Bruder SP, Jaiswal N, Ricalton NS, et al. Mesenchymal stem cells in osteobiology and applied bone regeneration. Clin Orthop Relat Res, 1998:S247-256.
79 Miyasaka M, Tanaka T. Lymphocyte trafficking across high endothelial venules: dogmas and enigmas. Nat Rev Immunol, 2004,4:360-370.
80 Wynn RF, Hart CA, Corradi-Perini C, et al. A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. Blood, 2004,104:2643-2645.
81 Von Luttichau I, Notohamiprodjo M, Wechselberger A, et al. Human adult CD34- progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4. Stem Cells Dev, 2005,14:329-336.
82 Sordi V, Malosio ML, Marchesi F, et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood, 2005,106:419-427.
83 Ji JF, He BP, Dheen ST, et al. Interactions of chemokines and chemokine receptors mediate the migration of mesenchymal stem cells to the impaired site in the brain after hypoglossal nerve injury. Stem Cells, 2004,22:415-427.
84 Kortesidis A, Zannettino A, Isenmann S, et al. Stromal-derived factor-1 promotes the growth, survival, and development of human bone marrow stromal stem cells. Blood, 2005,105:3793-3801.
85 Abbott JD, Huang Y, Liu D, et al. Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation, 2004,110:3300-3305.
86 Wang Y, Johnsen HE, Mortensen S, et al. Changes in circulating mesenchymal stem cells, stem cell homing factor, and vascular growth factors in patients with acute ST elevation myocardial infarction treated with primary percutaneous coronary intervention. Heart, 2006,92:768-774.
87 Wang L, Li Y, Chen J, et al. Ischemic cerebral tissue and MCP-1 enhance rat bone marrow stromal cell migration in interface culture. Exp Hematol, 2002,30:831-836.
88 Wang L, Li Y, Chen X, et al. MCP-1, MIP-1, IL-8 and ischemic cerebral tissue enhance human bone marrow stromal cell migration in interface culture. Hematology, 2002,7:113-117.
89 Batten P, Sarathchandra P, Antoniw JW, et al. Human mesenchymal stem cells induce T cell anergy and downregulate T cell allo-responses via the TH2 pathway: relevance to tissue engineering human heart valves. Tissue Eng, 2006,12:2263-2273.
90 Segers VF, Van Riet I, Andries LJ, et al. Mesenchymal stem cell adhesion to cardiac microvascular endothelium: activators and mechanisms. Am J Physiol Heart Circ Physiol, 2006,290:H1370-1377.
91 Schmidt A, Ladage D, Steingen C, et al. Mesenchymal stem cells transmigrate over the endothelial barrier. Eur J Cell Biol, 2006,85:1179-1188.
92 Devine SM, Bartholomew AM, Mahmud N, et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol, 2001,29:244-255.
93 Jiang WH, Ma AQ, Han K, et al. [Ultrastructural changes of rat ischemic myocardium after bone marrow mesenchymal stem cell transplantation]. Di Yi Jun Yi Da Xue Xue Bao, 2005,25:1090-1094.