三种底物对体外培养的神经干细胞分化和迁移能力的影响

目的:探讨不同底物对体外培养的神经干细胞分化和迁移能力的影响.方法:采用多聚鸟氨酸、层黏连蛋白、鼠尾胶原等作为底物,观察它们对大鼠神经干细胞分化的诱导和对细胞迁移能力的影响.结果:三种底物均能诱导神经干细胞的分化,诱导分化的能力为层黏连蛋白＞多聚鸟氨酸＞鼠尾胶原,多聚鸟氨酸与层黏连蛋白两者联用具有协同叠加效应,而且这些底物也具有介导分化后的神经细胞迁移的能力,各种底物对细胞迁移影响力的大小与其诱导分化的能力相似.结论:多聚鸟氨酸、层黏连蛋白和胶原不同程度上促进神经干细胞分化和迁移.     

不同底物对Sombati癫癎细胞模型整合素α2表达的影响

目的探讨不同底物对经体外培养的Sombati癫癎细胞模型海马神经元整合素α2表达水平的影响及意义。方法以层黏连蛋白和多聚L-赖氨酸作为底物于体外培养新生小鼠海马神经元,至第7天时行神经元纯度鉴定;无镁细胞外液继续培养3h后制备Sombati癫癎细胞模型,逆转录-聚合酶链反应检测不同处理组海马神经元整合素α2mRNA相对表达变化。结果模型制备前,经体外培养至第5天的层黏连蛋白组神经元胞体增大、饱满,突起连接紧密,神经网络形成,多聚L-赖氨酸组则体外培养至第7天时方出现上述表现;模型制备后24h,不同处理组神经元均可见迁移现象和神经网络“网格”样改变,但不同处理组神经元形态无明显差异。以整合素α2与β肌动蛋白光密度值之比值代表整合素α2mRNA相对表达量,多聚L．赖氨酸组、层黏连蛋白组和多聚L-赖氨酸＋层黏连蛋白组分别为0．25±0．03、0．37±0．05和0．48±0．09,后两组整合素α2mRNA表达水平高于多聚L-赖氨酸组,且差异有统计学意义（P=0．005,0．000）。结论与多聚L-赖氨酸组相比,层黏连蛋白对经体外培养的原代神经元贴壁及轴突连接具有较强的促进作用。添加外源性层黏连蛋白可诱导Sombati癫癎细胞模型海马神经元整合素α-mRNA表达上调。     

多聚赖氨酸和明胶对神经干细胞增殖和分化的影响

摘要 背景：目前大鼠神经干细胞体外诱导分化的研究报道诸多,但其分化过程很难控制,很多实验的操作方法复杂,分化比率也很低。 目的：探索大鼠胚胎前脑神经干细胞体外原代及传代培养方法,并观察其分化规律。 方法：胎鼠在无菌条件下分离出前脑,制备单细胞悬液,以1×1011L-1接种于含N2的DMEM/F12培养基中培养,传代培养过程中加入BrdU,标记神经干细胞球。诱导分化实验分为多聚赖氨酸铺板组、明胶铺板组和无铺板组。采用体积分数20%胎牛血清刺激其分化。免疫细胞化学检测nestin、BrdU及在血清诱导条件下神经干细胞向神经细胞分化的能力。 结果与结论：细胞呈神经干细胞样生长,具有连续增殖能力,可以传代培养。传代神经球中的细胞均呈nestin阳性和BrdU阳性。多聚赖氨酸铺板组和明胶铺板组贴壁后分化为神经细胞能力强于无铺板组(P < 0.01)。多聚赖氨酸铺板组略强于明胶铺板组(P > 0.05)。神经谱系标记物神经胶质纤维酸性蛋白和微管相关蛋白2的免疫细胞化学结果均阳性。结果表明,大鼠胚胎前脑富含神经干细胞,其分化观察,多聚赖氨酸和明胶在诱导神经干细胞分化中作为细胞贴壁支持物提高分化细胞数量的作用,且多分化为星形胶质细胞。 关键词：多聚赖氨酸;神经干细胞;明胶;增殖分化;体外培养 doi:10.3969/j.issn.1673-8225.2010.47.004     

肺癌细胞系中肿瘤干细胞的分离鉴定与功能分析*☆

背景：肿瘤干细胞在体外扩增和培养过程中如何保持其干细胞的特性成为肿瘤干细胞研究的重要障碍，从可长期稳定保存的细胞系中分离获得肿瘤干细胞可能成为肿瘤干细胞研究的可靠途径。 目的：探讨遗传背景相似、转移特性不同的肺癌细胞中肿瘤干细胞的存在与否及其功能。 方法：观察PLA-801D和PLA-801C细胞对鼠尾胶原和纤连蛋白的黏附作用，以考察两株细胞的转移潜能；观察PLA-801D和PLA-801C细胞形成单细胞克隆的种类及每种细胞克隆的亚克隆种类和性能，判断两株细胞中是否存在肿瘤干细胞；采用胶原收缩实验考察两株细胞的生物力学重塑能力，即对细胞外基质的重塑能力。 结果与结论：PLA-801D细胞对鼠尾胶原和纤连蛋白的黏附作用均明显强于PLA-801C细胞(P < 0.01)。PLA-801D可产生4种单细胞克隆，其中1种在传单细胞亚克隆后仍可形成如上4种克隆，而PLA-801C细胞中未发现有完全分化能力的细胞克隆。PLA-801D细胞的生物力学重塑能力也明显高于PLA-801C细胞(P < 0.05)。结果证实具有高转移潜能的PLA-801D细胞中含有一小群具有自我更新和分化能力的肿瘤干细胞，且该细胞株的生物力学重塑性较好，提示该细胞株对细胞外基质的重塑性能较强。     

不同底物对Sombati癫(癎)细胞模型整合素α2表达的影响

目的:探讨不同底物对经体外培养的Sombati癫(癎)细胞模型海马神经元整合素α2表达水平的影响及意义.方法:以层黏连蛋白和多聚L-赖氨酸作为底物于体外培养新生小鼠海马神经元,至第7天时行神经元纯度鉴定;无镁细胞外液继续培养3h后制备Sombati癫(癎)细胞模型,逆转录-聚合酶链反应检测小同处理组海马神经元整合素α2...     

胚胎大鼠纹状体神经干细胞的体外培养和分化

目的 :旨在探讨纹状体神经干细胞 (striatum neuralstemcells ,strNSCs)的培养及分化鉴定方法。方法 :选择性分离纹状体的神经干细胞 ,体外培养、扩增和诱导分化 ,并采用免疫荧光细胞化学检查鉴定。结果 :从胚鼠纹状体分离的细胞具有连续克隆能力 ,绝大多数细胞表达巢蛋白。诱导分化后的细胞表达成熟神经细胞和星形胶质细胞特异性的蛋白。结论 :用此方法分离的细胞具有自我更新、增殖和分化潜能 ,具备中枢神经系统干细胞的一般特征。     

大鼠胎脑皮质神经干细胞体外培养及诱导分化的实验研究

目的 从孕龄15d SD胚胎鼠脑皮质中分离并培养神经干细胞（neural stem cells。NSCs），观察其生长、增殖及分化。方法 采用包含碱性成纤维细胞生长因子（bFGF）和表皮细胞生长因子（EGF）的无血清培养及单细胞克隆技术，对胚胎鼠脑皮质神经干细胞进行原代、传代培养及诱导其分化。用Nestin染色鉴定神经干细胞特性，用免疫组化方法（β-Ⅲ-tubulin、GFAP染色）检测神经干细胞分化为神经元及神经胶质细胞状况。结果 从孕龄15dSD胚胎鼠脑皮质中分离的组织，经原代及传代培养均可形成细胞克隆．切具有增殖能力。原代及传代培养细胞呈Nestin（神经上皮干细胞蛋白）表达阳性．诱导分化后的细胞表达神经元细胞、星形胶质细胞的特异性抗原。结论 本实验分离、培养的孕龄15dSD胚胎鼠脑皮质细胞Nestin表达阳性．分化后表达神经元和星形胶质细胞的标记物，是大鼠的神经干细胞，并具有多向分化潜能。     

早期分化的神经干细胞移植治疗大鼠脑梗死的实验研究

目的研究早期分化的神经干细胞移植治疗脑梗死的可能性。方法从Wistar新生大鼠的大脑分离培养神经干细胞,取传代的神经干细胞诱导分化并经BrdU(5-溴脱氧尿嘧啶)标记后移植到脑梗死对侧的侧脑室,移植后对大鼠的功能恢复进行评价,用免疫组织化学方法鉴定移植的细胞在脑内的迁移和分化情况。结果将早期分化的神经干细胞移植到鼠脑后2周在梗死灶对侧可发现移植的细胞,4周时移植的细胞在梗死灶内分化为神经细胞,大鼠的学习功能和神经功能恢复较对照组均有明显改善。结论早期分化的神经干细胞移植到大鼠脑内仍能存活,并能有效穿过脑脊液———脑屏障迁移到脑梗死的部位;且分化为神经细胞。     

不同体外诱导培养条件下新生鼠基底前脑神经干细胞增殖和分化为神经元的能力*☆

目的：研究神经干细胞的增殖、迁移和分化可为揭示神经系统的发生、发育过程提供可靠依据。观察表皮生长因子和碱性成纤维生长因子在体外刺激新生鼠基底前脑神经干细胞的增殖情况，及其各自诱导神经干细胞分化成神经元的能力。 方法：实验于2005-12/2006-07在广州医学院解剖学教研室完成。①动物：清洁级新生24 h内的SD大鼠30只，实验过程中对动物的处置符合动物伦理学标准。②实验方法：新生鼠在无菌条件下取脑，分离出基底前脑，胰蛋白酶消化，离心过滤制备单细胞悬液，接种于含B27的DMEM/F12培养基培养瓶中，每瓶40~60万个细胞，加入终浓度均为10μg/L的表皮生长因子和碱性成纤维生长因子刺激生长，在体外进行神经干细胞的克隆培养，传代培养过程中加入终浓度为6 mg/L BrdU用于标记神经球，设立3组，各自加入体积分数为0.1的小牛血清、终浓度均为10μg/L的表皮生长因子、碱性成纤维生长因子，对培养得到的神经干细胞进行诱导分化。③实验评估：免疫荧光染色检测神经干细胞巢蛋白抗原的表达，并用BrdU标记和免疫荧光证实其增殖能力。免疫荧光染色检测不同诱导条件下神经干细胞向神经元分化的能力。 结果：①细胞形态观察：从新生鼠基底前脑成功分离出神经干细胞，原代培养呈透亮的圆球形，2~3 d后细胞数目明显减少，部分细胞开始分裂。1周左右培养瓶中出现许多由数十到数百个细胞组成的悬浮生长的细胞球，球中的细胞形态规则，边界清楚，折光性较强，胞浆颜色较深，核/浆比较大。该细胞具有连续增殖能力，可以传代培养。②巢蛋白抗原的表达：传代神经球中的细胞均呈巢蛋白抗原阳性。③BrdU标记检测：克隆球中的细胞均为BrdU阳性，表明克隆球是由不断分裂增殖的细胞组成。④诱导分化结果：在体积分数为0.1小牛血清、终浓度均为10μg/L的表皮生长因子、碱性成纤维生长因子诱导条件下，神经干细胞分化为神经元的比例分别为20%，22%，40%。 结论：①表皮生长因子和碱性成纤维生长因子在体外能够刺激新生鼠基底前脑神经干细胞连续增殖，且具有胚胎源性。②以血清作为对照，碱性成纤维生长因子诱导神经干细胞向神经元分化的能力强于表皮生长因子。     

成年C57小鼠脊髓神经干细胞的培养与鉴定

目的　 从成年小鼠脊髓中培养神经干细胞 ,并对其进行鉴定和诱导分化。方法　 成年C5 7小鼠 ,悬浮培养神经干细胞技术。结果　培养 1～ 2周时 ,培养液中即可出现神经干细胞克隆球。该克隆球具有很强的自我增殖能力 ,可多次传代。免疫细胞化学技术证明该克隆球表达大量的神经干细胞特征性的中间丝———巢蛋白(Nestin) ;经 1%胎牛血清诱导后可表达神经元、星形胶质细胞和少突胶质细胞特征性标志物β tubulinIII、胶质纤维酸性蛋白 (Gliafibrillaryacidicprotein ,GFAP)和RIP ,提示它们可朝神经元、星形胶质细胞和少突胶质细胞方向分化。 结论　 本研究结果提示正常成年C5 7小鼠脊髓中含有神经干细胞 ,在体外条件下可以大量增殖 ,经诱导后可朝神经元和胶质细胞的方向分化     

Sphingosine 1-phosphate-primed astrocytes enhance differentiation of neuronal progenitor cells

Sphingosine 1‐phosphate (S1P) is a bioactive signaling lysophospholipid. Effects of S1P on proliferation, survival, migration, and differentiation have already been described; however, its role as a mediator of interactions between neurons and glial cells has been poorly explored. Here we describe effects of S1P, via the activation of its receptors in astrocytes, on the differentiation of neural progenitor cells (NPC) derived from either embryonic stem cells or the developing cerebral cortex. S1P added directly to NPC induced their differentiation, but S1P‐primed astrocytes were able to promote even more pronounced changes in maturation, neurite outgrowth, and arborization in NPC. An increase in laminin by astrocytes was observed after S1P treatment. The effects of S1P‐primed astrocytes on neural precursor cells were abrogated by antibodies against laminin. Together, our data indicate that S1P‐treated astrocytes are able to induce neuronal differentiation of NPC by increasing the levels of laminin. These results implicate S1P signaling pathways as new targets for understanding neuroglial interactions within the central nervous system. © 2012 Wiley Periodicals, Inc.     

Influence of culture substrata on the differentiation of advanced passage glial cells in cultures from aged mouse cerebral hemispheres

We have previously reported that glial cells derived from aged mouse cerebral hemispheres (MACH) in primary cultures and after several passages consist of protoplasmic astrocytes (Type 1), differentiated stellate astrocytes (Type 2), a few oligodendrocytes and also glial precursors.^4,33,34 In this study, we examined the influence of culture substrata: plastic, poly-l-lysine, laminin or collagen on the differentiation of MACH glial cells of advanced passages (P18–19) using glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP) activity as biochemical markers for astrocytes and oligodendrocytes, respectively. Cultures were also examined morphologically using light microscopy. In general, GS activity was increased in cultures grown on the three chemical substrata versus plastic alone with the most striking effect being the 2-fold increase observed in those cells grown in laminin. No differences were noted in CNP activity. Morphologically, proliferation of protoplasmic (Type 1) astrocytes was enhanced by culture day 2 on polylysine substratum and stellate differentiated (Type 2) astrocytes were noted on collagen. The striking feature in cultures grown on laminin was the presence of astrocytes with markedly long processes. Thus, morphological astrocyte differentiation appears to correspond to the increased GS activity. We propose that the extracellular matrix components such as collagen and laminin may play an important role in promoting glial precursors to differentiate into astrocytes.     

Surface topography during neural stem cell differentiation regulates cell migration and cell morphology

We sought to determine the contribution of scaffold topography to the migration and morphology of neural stem cells by mimicking anatomical features of scaffolds found in vivo. We mimicked two types of central nervous system scaffolds encountered by neural stem cells during development in vitro by constructing different diameter electrospun polycaprolactone (PCL) fiber mats, a substrate that we have shown to be topographically similar to brain scaffolds. We compared the effects of large fibers (made to mimic blood vessel topography) with those of small‐diameter fibers (made to mimic radial glial process topography) on the migration and differentiation of neural stem cells. Neural stem cells showed differential migratory and morphological reactions with laminin in different topographical contexts. We demonstrate, for the first time, that neural stem cell biological responses to laminin are dependent on topographical context. Large‐fiber topography without laminin prevented cell migration, which was partially reversed by treatment with rock inhibitor. Cell morphology complexity assayed by fractal dimension was inhibited in nocodazole‐ and cytochalasin‐D–treated neural precursor cells in large‐fiber topography, but was not changed in small‐fiber topography with these inhibitors. These data indicate that cell morphology has different requirements on cytoskeletal proteins dependent on the topographical environment encountered by the cell. We propose that the physical structure of distinct scaffolds induces unique signaling cascades that regulate migration and morphology in embryonic neural precursor cells. J. Comp. Neurol. 524:3485–3502, 2016. © 2016 Wiley Periodicals, Inc.     

Novel nanometer scaffolds regulate the biological behaviors of neural stem cells

Ideal tissue-engineered scaffold materials regulate proliferation, apoptosis and differentiation of cells seeded on them by regulating gene expression. In this study, aligned and randomly oriented collagen nanofiber scaffolds were prepared using electronic spinning technology. Their diameters and appearance reached the standards of tissue-engineered nanometer scaffolds. The nanofiber scaffolds were characterized by a high swelling ratio, high porosity and good mechanical properties. The proliferation of spinal cord-derived neural stem cells on novel nanofiber scaffolds was obviously enhanced. The proportions of cells in the S and G 2 /M phases noticeably increased. Moreover, the proliferation rate of neural stem cells on the aligned collagen nanofiber scaffolds was high. The expression levels of cyclin D1 and cyclin-dependent kinase 2 were increased. Bcl-2 expression was significantly increased, but Bax and caspase-3 gene expressions were obviously decreased. There was no significant difference in the differentiation of neural stem cells into neurons on aligned and randomly oriented collagen nanofiber scaffolds. These results indicate that novel nanofiber scaffolds could promote the proliferation of spinal cord-derived neural stem cells and inhibit apoptosis without inducing differentiation. Nanofiber scaffolds regulate apoptosis and proliferation in neural stem cells by altering gene expression.     

Wnt-3a protein promote neuronal differentiation of neural stem cells derived from adult mouse spinal cord

BACKGROUND: Wnt proteins as growth factor have multiple functions in neural development, and especially serve key roles in differentiation and development. Wnt-3a is an intercellular signaling molecule that is involved in a variety of morphogenetic events. The purpose of this study was to investigate the effects of Wnt-3a signal protein on proliferation and differentiation of neural stem cells derived from adult mouse spinal cord. METHODS: Adult mouse neural stem cells were cultured with serum free incubation. The recombined plasmid pSecTag2/Hygro B-Wnt3a for eukaryotic expression transfected adult neural stem cell, then the expression protein was detected by Western blot. The differentiation of adult neural stem cells was identified by the immunocytochemical technique. RESULTS: The inducing differentiated rates of neurons were improved greatly by Wnt-3a protein compared with control (p<0.05). CONCLUSION: Wnt-3a has obvious influence on the neuronal differentiation of adult neural stem cell.     

Regulation of human neural precursor cells by laminin and integrins

Deciphering the factors that regulate human neural stem cells will greatly aid in their use as models of development and as therapeutic agents. The extracellular matrix (ECM) is a component of stem cell niches in vivo and regulates multiple functions in diverse cell types, yet little is known about its effects on human neural stem/precursor cells (NSPCs). We therefore plated human NSPCs on four different substrates (poly-L-ornithine, fibronectin, laminin, and matrigel) and compared their responses with those of mouse NSPCs. Compared with the other substrates, laminin matrices enhanced NSPC migration, expansion, differentiation into neurons and astrocytes, and elongation of neurites from NSPC-derived neurons. Laminin had a similar spectrum of effects on both human and mouse cells, highlighting the evolutionary conservation of NSPC regulation by this component of the ECM. Flow cytometry revealed that human NSPCs express on their cell surfaces the laminin-binding integrins alpha3, alpha6, alpha7, beta1, and beta4, and function-blocking antibodies to the alpha6 subunit confirmed a role for integrins in laminin-dependent migration of human NSPCs. These results define laminin and its integrin receptors as key regulators of human NSPCs.     

Time-sensitive effects of hypoxia on differentiation of neural stem cells derived from mouse embryonic stem cells in vitro

Abstract

Objectives:

Oxygen tension is an important component of microenvironment for the differentiation of embryonic stem cells including neural lineage. However, the comprehensive influence of hypoxia on neural differentiation during embryonic neural development has not yet been examined.

Methods:

In this study, we investigated the effect of low oxygen levels (5% O₂), or hypoxia, in two stages of neural differentiation in vitro: (1) inducing mouse embryonic stem cells into neural stem cells (NSCs); and then (2) inducing NSCs into neural progenitor cells in neurospheres.

Results:

In the first stage, NSCs generation was reduced under hypoxia. Less mature morphological changes (including neural marker) of NSCs were observed, suggesting the prevention of early differentiation under hypoxic conditions. Thus undifferentiated stem cells were maintained in this stage. However, in the second stage, hypoxia induced neural differentiation in neurospheres. Nevertheless, non-neural progenitor cell formation, such as mesoderm progenitor cell lines or epithelial cell lines, was restricted by low oxygen tension.

Discussions:

Our results demonstrate that hypoxia is essential for regulating neural differentiation and show the different effects on NSC differentiation dependent on the time-course of NSC development. In the early stage of NSCs induction, hypoxia inhibits neural differentiation and maintains the undifferentiated state; in the later stage of NSCs induction, hypoxia induces neural differentiation. Our study may contribute to the development of new insights for expansion and control of neural differentiation.     

4002/大鼠海马中83KD蛋白诱导神经干细胞向AChE阳性神经元定向分化的作用（英文）

背景：神经干细胞的临床应用还尚待时日,现阶段需要解决如何诱导神经干细胞分化为特定表型的神经元以替代丢失、变性的神经元细胞。 目的：探讨大鼠海马组织83 ku蛋白对神经干细胞向乙酰胆碱酯酶阳性神经元分化的作用。 设计、时间及地点：细胞学体外对照观察,于2003-10/2008-04在南通大学医学院完成。 材料：清洁级SD大鼠12只,17 d龄SD胎鼠多只,均由南通大学实验动物中心提供。 方法：取6只正常大鼠及6只切割海马伞后14 d大鼠的海马组织制成匀浆,进行非变性聚丙烯酰胺凝胶电泳,根据染色结果切取含有83 ku差异蛋白条带进行电洗脱,定量后调整蛋白浓度为300 mg/L。取胎鼠前脑组织,体外分离培养神经干细胞,设立3组：空白对照组加入单纯DMEM/F12无血清培养基;83 ku蛋白正常组、83 ku蛋白切割组分别加入含10 mg/L来自正常/割海马伞大鼠海马组织83 ku蛋白的DMEM/F12无血清培养基,诱导12 d。 主要观察指标：用乙酰胆碱酯酶组织化学染色检测神经干细胞分化为乙酰胆碱酯酶阳性神经元的情况。 结果：诱导12 d后,83 ku蛋白切割组乙酰胆碱酯酶阳性神经元较多,胞体大且分化较好,突起粗且长;83 ku蛋白正常组乙酰胆碱酯酶阳性神经元较少,胞体小,突起短;空白对照组仅见少量乙酰胆碱酯酶阳性神经元。组间乙酰胆碱酯酶阳性神经元数比较差异有显著性意义(P < 0.05),83 ku蛋白切割组>83 ku蛋白正常组>空白对照组。 结论：大鼠海马组织中83 ku蛋白可成功诱导神经干细胞定向分化为乙酰胆碱酯酶阳性神经元。     

Wnt-3a protein promote neuronal differentiation of neural stem cells derived from adult mouse spinal cord

Abstract

Background: Wnt proteins as growth factor have multiple functions in neural development, and especially serve key roles in differentiation and development. Wnt-3a is an intercellular signaling molecule that is involved in a variety of morphogenetic events. The purpose of this study was to investigate the effects of Wnt-3a signal protein on proliferation and differentiation of neural stem cells derived from adult mouse spinal cord.

Methods: Adult mouse neural stem cells were cultured with serum free incubation. The recombined plasmid pSecTag2/Hygro B-Wnt3a for eukaryotic expression transfected adult neural stem cell, then the expression protein was detected by Western blot. The differentiation of adult neural stem cells was identified by the immunocytochemical technique.

Results: The inducing differentiated rates of neurons were improved greatly by Wnt-3a protein compared with control (p<0.05).

Conclusion: Wnt-3a has obvious influence on the neuronal differentiation of adult neural stem cell.     

Cell attachment and neurite stability in NG108-15 cells: effects of 5'-deoxy, 5'-methyl thioadenosine (MTA) compared with laminin, kinase inhibitor H-7, and Mn2+ ions

Methylation inhibitors such as 5'-deoxy, 5'-methyl thioadenosine (MTA) have been shown to exert an intriguing spectrum of effects upon neural cells: inhibiting responses to nerve growth factor in PC12 cells; stimulating outgrowth of rapid-onset neurites in NG108-15 cells; inhibiting the resorption and remodelling of these rapid-onset neurites; and inducing fibroblasts to extend long, multipolar, branched processes. To learn whether the apparently diverse effects of these agents might reflect some common underlying cellular sites of action, we examined simpler, short-term effects of MTA upon cell attachment and stability of rapid-onset neurites in NG108-15 cells, and compared the effects with those produced by the kinase inhibitor H-7, substratum-bound laminin, or Mn2+ ions. MTA was shown to selectively enhance the response of cells and their neurites in attaching to their substrata, without inducing other 'adhesive' responses such as cell spreading or motility. The data suggest that MTA activates or increases the effectiveness of 'attachment receptors', and that this at least partially explains its neurite-promoting effects. While it is still premature to propose a common mechanism of action for MTA and related agents, all of their known effects thus appear to involve modulating responses generated at cell-surface receptors. A further clue is that kinase C activity appears to be critical, since cells pretreated with phorbol esters for 24 h (which down-regulated levels of kinase C) selectively failed to extend neurites in response to MTA, but responded normally to laminin and Mn2+ ions.