体外诱导大鼠骨髓间充质干细胞向内耳毛细胞样细胞分化的研究

目的 探讨体外定向诱导大鼠骨髓间充质干细胞向耳蜗毛细胞样细胞分化的可行性.方法 分离培养大鼠骨髓间充质干细胞,并加以鉴定.分离出生1～3 d的乳鼠耳蜗Corti器,与骨髓间充质干细胞在体外共培养14 d.通过反转录聚合酶链反应(RT-PCR)和免疫细胞化学染色对分化细胞的特异性分子指标(myosinⅦa、math1及calretinin)进行鉴定.结果 免疫细胞化学染色显示诱导分化后的细胞myosinⅦa、math1和calretinin表达阳性,RT-PCR提示分化后的细胞可表达毛细胞的标志物myosinⅦa和math1.结论 体外培养的骨髓间充质干细胞可诱导分化为具有毛细胞分子标志物的毛细胞样细胞.     

体外诱导人脐带间充质干细胞向内耳毛细胞样细胞分化的研究

目的 探讨并建立一种体外定向诱导人脐带间充质干细胞(human umbilical cord mesenchymal stem cells ,hUC-MSCs)向内耳毛细胞样细胞分化的方法.方法 采用组织贴壁法培养获得hUC-MSCs,流式细胞仪鉴定其表面标记物;神经干细胞诱导阶段分为对照组(DFNB基础诱导培养基)、实验1组(DFNB基础诱导培养基+20 ng/ml EGF+20 ng/ml bFGF)、实验2组(DFNB基础诱导培养基+20 ng/ml EGF+20 ng/ml bFGF+琼脂糖包被),培养3~5天后采用免疫荧光法检测各组标记蛋白Nestin的表达;内耳毛细胞样细胞诱导分化阶段,分为对照组(DFNB基础诱导培养基+明胶包被)、诱导组[DFNB基础诱导培养基+20 ng/ml EGF+1 μM全反式维甲酸/全反式维生素A酸(all trans retinoic aid,ATRA)],培养4周后采用qRT-PCR和免疫荧光法检测各组毛细胞标记物Math1、MyosinⅦa、Brn3c的表达.结果 在神经干细胞诱导阶段,对照组、实验1组均无神经球结构,实验2组中hUC-MSCs分化3 d后细胞有明显神经球样结构,且Nestin阳性细胞数更高(P<0.05);而向内耳毛细胞样诱导分化4周后,诱导组毛细胞标记物Math1、MyosinⅦa、Brn3c的mRNA和阳性细胞数的表达水平明显升高(P<0.05).结论 体外诱导人脐带间充质干细胞先向神经干细胞方向分化再向内耳毛细胞分化方向诱导,可有效提高毛细胞标记物MyosinⅦa、Brn3c、Math1的表达水平,促进hUC-MSCs向类毛细胞样细胞分化,可能为将来感音神经性聋的内耳移植治疗提供更为适合的种子细胞.     

iPSCs定向分化的内耳毛细胞与支持细胞间相互作用的研究

目的利用诱导多能性干细胞定向分化的内耳毛细胞和支持细胞探究这两种体外诱导分化细胞之间的相互作用。方法首先,利用细胞单层贴壁两步诱导法将三株i PS细胞(野生株、MYO7A缺陷株、MYO7A校正株)向内耳祖细胞及内耳毛细胞诱导分化,探究i PSCs定向分化内耳毛细胞的过程中是否有支持细胞的产生;其次,通过细胞免疫化学的方法探究体外分化的内耳毛细胞和支持细胞间的相互作用;最后,将表达绿色荧光蛋白(EGFP)的上皮样内耳祖细胞以圆窗膜穿刺的方法移植到白化荣昌猪的内耳中观察分析移植细胞在体内的迁移、分化以及在体内形成的联系。结果三株i PS细胞诱导分化为内耳毛细胞的过程中均有一部分细胞分化为支持细胞;对分化细胞进行E-cadherin、N-cadherin和ZO-1的免疫荧光检测结果显示,E-cadherin、N-cadherin和ZO-1在支持细胞-支持细胞连接和支持细胞-毛细胞连接间都有表达;移植4周后,耳蜗免疫组织化学结果显示,三株不同来源的移植细胞均有少量细胞成功迁移到了毛细胞受损部位—柯底氏器,并表达毛细胞标志性蛋白MYO7A。移植细胞之间以及移植细胞与宿主细胞之间有E-cadherin、N-cadherin和ZO-1的表达。结论 i PSCs诱导分化的内耳毛细胞和支持细胞在体内、外均能形成钙粘连接和紧密连接。这些研究结果对毛细胞取代法治疗耳聋策略的完善与发展有一定的科学意义。     

新生豚鼠海马神经干细胞分化为类毛细胞的体外实验

目的体外定向诱导新生豚鼠海马神经干细胞分化为内耳毛细胞。方法将原代培养的新生豚鼠海马来源的神经干细胞进行体外培养,分别置入含有10%胎牛血清和5%～15%人工外淋巴液培养基使其分化,并用免疫荧光化学,蛋白免疫印迹扫描电镜对分化的细胞进行鉴定。结果胎牛血清和人工外淋巴液均能诱导新生豚鼠海马神经干细胞分化为表达毛细胞特异抗体的细胞。结论神经干细胞在胎牛血清和人工外淋巴液中可以存活并分化出表达毛细胞特异抗体的细胞,为神经干细胞移植内耳治疗提供理论依据。     

胎鼠神经干细胞的原代培养及体外诱导分化研究

目的探讨神经干细胞的原代培养以及体外诱导分化为毛细胞样细胞的方法 ,为神经干细胞移植治疗感音神经性聋奠定基础。方法取胎龄为14.5天的SPF级昆明小鼠脑组织,采用机械吹打的方法在无血清培养基中原代培养,传代培养,诱导分化为毛细胞样细胞,采用免疫细胞化学方法鉴定神经干细胞以及毛细胞的免疫表型。结果①在含成纤维细胞生长因子-2（FGF-2）和表皮生长因子（EGF）的无血清培养液中,神经干细胞在体外培养6～8代后,其细胞呈指数级增加,增殖的细胞表达神经上皮干细胞蛋白（nestin）;②在诱导分化培养基中培养3周后它们能被诱导分化为神经胶质细胞、毛细胞样细胞,4周后表达支持细胞表型。结论神经干细胞在体外可以诱导分化为毛细胞样细胞。     

Atoh1基因与内耳毛细胞再生

哺乳动物毛细胞的产生和内耳形态的生成是由局部细胞间相互影响以及特异的基因所调控.Atoh1基因是内耳感觉细胞分化过程中的重要正性调控因子,Atoh1的过度表达可产生新的具有感觉功能的毛细胞,且具有吸引神经轴突的能力.本文对Atoh1基因与细胞诱导分化为内耳毛细胞的研究进行综述.     

Atoh1基因与内耳毛细胞再生

哺乳动物毛细胞的产生和内耳形态的生成是由局部细胞间相互影响以及特异的基因所调控.Atoh1基因是内耳感觉细胞分化过程中的重要正性调控因子,Atoh1的过度表达可产生新的具有感觉功能的毛细胞,且具有吸引神经轴突的能力.本文对Atoh1基因与细胞诱导分化为内耳毛细胞的研究进行综述.     

羊水干细胞拟胚体与耳蜗基底膜共培养向神经元分化的可行性

目的探讨人来源的羊水干细胞以拟胚体样结构与小鼠耳蜗基底膜组织共培养,分化为神经元的可行性。方法分离培养人来源的羊水干细胞,悬滴法培养,观察羊水干细胞拟胚体形成情况;免疫荧光检测拟胚体细胞干性标记物的表达;将拟胚体与小鼠基底膜组织共培养,不添加神经生长因子及神经元信号诱导因子,观察拟胚体向神经元分化情况。结果羊水干细胞经悬滴培养可形成拟胚体样结构,表达神经干细胞标记物Sox2、Nestin;与小鼠耳蜗基底膜组织共培养,拟胚体细胞有神经元标记物Tuj1表达,但无神经元形态特征。结论羊水干细胞体外悬滴后可形成形态均一且具有神经干性的拟胚体,与耳蜗基底膜组织共培养,不能诱导拟胚体细胞向形态典型的神经元分化。     

来自于胚胎和诱导多能干细胞的机械感受性毛细胞样细胞

Olaima等研究者应用体外细胞培育技术,将小鼠胚胎干细胞(embryonic stem cells,ESCs)和诱导多能干细胞(induced pluripotent stem cells,iPSCs)转化成为成群的耳祖细胞(otic progenitor cells),并进而分化为机械感受性内耳毛细胞样细胞(hair cell-like cells).     

诱导新生大鼠耳蜗大上皮嵴细胞分化为毛细胞样细胞

目的大鼠耳蜗大上皮嵴细胞（greater epithelial ridge,GER）转染Hath1基因,与耳蜗间质细胞共培养诱导分化为毛细胞样细胞。方法取出生后第1天的大鼠耳蜗,利用机械分离和酶消化相结合的方法分离出纯的GER细胞,与耳蜗间质细胞共培养或转染Hath1基因培养,做免疫组化和扫描电镜观察。结果GER体外培养具有增殖能力,GER与耳蜗间质细胞共培养或转染Hath1基因后,免疫组化MyosinVIIa阳性,扫描电镜部分细胞的表面可见不成熟的纤毛样结构,提示GER已分化为毛细胞样细胞。结论GER细胞作为毛细胞前体细胞可以实现体外培养,与耳蜗间质细胞共培养或转染Hath1后,可分化为毛细胞样细胞。     

Fates of mouse embryonic stem cells transplanted into the inner ears of adult mice and embryonic chickens

The potential of embryonic stem (ES) cells to differentiate into inner ear hair cells was examined in this study. Undifferentiated mouse ES cells transplanted into neomycin-damaged mouse inner ears were evaluated by immunohistochemistry 4 weeks after transplantation. Some ES cells were positive for E-cadherin or NCAM, and most transplanted cells were positive for SSEA3 and Ki67. None were positive for Myosin VIIa or MF20. These results indicate that the damaged inner ear may have some activity inducing ES cells to develop into ectoderm cells, but the effect was insufficient to induce inner ear hair cells. Next, SDIA/BMP-treated ES cells were transplanted into embryonic chicken inner ear rudiments. Embryonic chickens were expected to share the same developmental systems as mice. SDIA/BMP treatment drove ES cells to the population including neural crest cells and probably placode cells ES colonies were found next to or in the otic vesicles but were not a part of vesicle walls, indicating that transplanted ES cells could not be expected to be the same kind of cells as chicken otic vesicle cells Some ES colonies were found at the vestibulo-cochlear ganglions. To induce inner ear hair cells in this system, the competency of ES cells and otic induction signals should be defined further.     

Retinoic acid induces differentiation of cochlear neural progenitor cells into hair cells

IntroductionInner ear progenitor cells have the potential for multi-directional differentiation. Retinoic acid is an important requirement for the development of the inner ear. Blocking the Curtyr's retinoic acid signaling pathway can significantly reduce the number of hair cells. Therefore, we believe that retinoic acid may induce the regeneration of inner ear hair cells.ObjectiveTo investigate whether the cochlear neural progenitor cells maintain the characteristics of stem cells during recovery and subculture, whether retinoic acid can induce cochlear neural progenitor cells into hair cells in vitro, and whether retinoic acid promotes or inhibits the proliferation of cochlear neural progenitor cells during differentiation.MethodsCochlear neural progenitor cells were cultured and induced in DMEM/F12 + RA (10^?6 M) and then detected the expressions of hair cell markers (Math1 and MyosinVIIa) by immunofluorescence cytochemistry and realtime-polymerase chain reaction, and the proliferation of cochlear neural progenitor cells was detected by Brdu.ResultsThe nestin of cochlear neural progenitor cells was positively expressed. The ratios of Math1-positive cells in the control group and experimental group were 1.5% and 63%, respectively; the ratios of MyosinVIIa-positive cells in the control group and experimental group were 0.96% and 56%, respectively (p < 0.05). The ratios of Brdu⁺-labeled cells in retinoic acid group, group PBS, and group FBS were 20.6%, 29.9%, and 54.3%, respectively; however, the proliferation rate in the experimental group decreased.ConclusionRetinoic acid can promote cochlear neural progenitor cells to differentiate into the hair cells.     

Generation of inner ear hair cell immunophenotypes from neurospheres obtained from fetal rat central nervous system in vitro

Neural stem cells are suggested to possess a highly plastic ability to differentiate into several specific cell types, not only neuronal lineages but also other germ layer tissue-specific cell lineages. To examine whether hair cell immunophenotypes could be derived from the central nervous system (CNS), we established cell cultures from embryonic day 16.5 fetal rat brain tissues, and analyzed changes in immunohistochemical features of the CNS cell cultures by induction of differentiation. The results of this study showed that neural progenitors obtained from fetal rat CNS generated hair cell immunophenotypes with expression of both epitopes of hair cell marker proteins Brn-3c and myosin VIIa in vitro. These findings indicate that immature neural progenitors possess the potential to differentiate into hair cell phenotypes. Immature neural progenitors may be useful as materials for cell transplantation therapy for replacement of damaged inner ear hair cells.     

胚胎大鼠神经干细胞体外培养及分化为类毛细胞的研究

目的探讨体外培养和鉴定胚胎大鼠神经干细胞(neural stem cells,NSCs)及诱导其分化为类毛细胞。方法从SD系胚鼠大脑分离NSCs,在无血清培养基中培养,用含10%胎牛血清的DMEM/F12培养基诱导其分化为神经元和星形胶质细胞:将NSCs球放到含鼠尾胶原包被的盖玻片6孔培养板中培养,然后将乳鼠基底膜体外培养后汲取其上清液与NSCs一起培养,14～21天后通过免疫荧光和免疫组化法检测毛细胞标志物肌球蛋白(myosin)Ⅶa和钙视网膜蛋白(calretinin)。结果培养的NSCs胞体透亮,折光性好,分化后的细胞免疫组化示神经元特异性烯醇化酶、胶原纤维酸性蛋白、myosinⅦa和calretinin阳性。结论在无血清条件下能培养出活性很好的NSCs,并且能诱导其分化为神经元、星形胶质细胞和类毛细胞。     

Effects of bone morphogenetic protein 4 on differentiation of embryonic stem cells into myosin VIIa-positive cells

CONCLUSION: Our results indicate that myosin VIIa-positive cells are generated from embryonic stem cells (ESCs) co-cultured with PA6 cells; however, bone morphogenetic protein 4 (BMP4) may not be a key molecule for induction of myosin VIIa-positive cells from the ESCs. BACKGROUND: ESCs have been considered as a basis for cell therapy in a range of organs, because of their potential for self-renewal and pluripotency. Co-culture with PA6 stromal cells can induce differentiation of ESCs into various types of ectodermal cells including sensory progenitors. BMP4 plays an essential role in the development of sensory hair cells in the inner ear. MATERIALS AND METHODS: We examined effects of BMP4 on differentiation of ESCs into the hair cell immunophenotype. BMP4 was supplemented at different time points to ESCs co-cultured on PA6 stromal cells. The ESCs were then collected and examined for the expression of myosin VIIa, a hair cell marker, and betaIII-tubulin, a neural marker. The expression of myosin VIIa and betaIII-tubulin was identified. RESULTS: Quantitative assessments revealed that exogenous BMP4 has significant effects on the expression of betaIII-tubulin, but not of myosin VIIa.     

Development and regeneration of hair cells

The vertebrate inner ear is derived from the otic placode and undergoes a complicated series of morphogenetic processes to differentiate into an elaborate structure harboring mechanosensory epithelia featuring hair cells, the mechanoreceptors of hearing and balance. Recently, the principal mechanisms producing hair cells and the key molecules involved in their fate determination and differentiation have been gradually unveiled. The in-depth understanding of hair cell development is consequently providing clues to strategies for mammalian hair cell regeneration. Among them, the identification and characterization of progenitor cells for the hair cell lineage, which is just emerging, is of particular interest. Herein, we review the molecular mechanisms of inner ear development with particular focus on perspectives for hair cell regeneration.     

Expression of calretinin by fetal otocyst cells after transplantation into damaged rat utricle explants

Severe damage by acoustic overstimulation or ototoxins induces inner ear hair cell loss, resulting in permanent hearing loss and balance disorders because hair cell regeneration scarcely occurs in the inner ear sensory organs of mammals. In this study, to evaluate the possibilities of cell transplantation therapy for damaged inner ear sensory organs, dissociated cell cultures of fetal otocyst cells (FOCs) were established from embryonic day 12.5 (E12.5) rat inner ears, and transplanted into gentamicin-treated explants of vestibular sensory epithelia. Two weeks after transplantation, immunohistochemical analysis demonstrated that some of the grafted FOCs survived within the vestibular sensory epithelia and expressed epitopes of calretinin. one of the hair cell marker proteins. These findings indicate that FOCs have the potential to migrate into damaged vestibular epithelia and differentiate into hair cell immunophenotypes. Cell transplantation therapy may be available for functional regeneration in inner ear diseases.     

成人鼻黏膜间充质干细胞的体外培养和多向诱导分化

目的:观察成人鼻腔呼吸黏膜内间充质干细胞(MSCs)的分布特征,建立鼻黏膜MSCs的体外培养方法,探讨该细胞向神经细胞及成骨细胞分化的潜能。方法:将成人鼻腔呼吸黏膜制成冷冻切片,用Nestin和Vi-mentin抗体进行免疫荧光染色,观察MSCs在鼻黏膜内的分布特征。对鼻黏膜细胞进行体外培养、传代,扩增出MSCs,用干细胞标志蛋白Nestin,CD133,Vimentin和Sall4的抗体进行免疫荧光染色,鉴定该细胞的干细胞特性;分别用成骨诱导培养液(含地塞米松、维生素C和β-甘油磷酸钠)及神经诱导培养液(Neurobasal培养液含B27,ATRA,TSA)诱导MSCs向成骨细胞和神经细胞定向分化;用组织化学染色和免疫荧光染色方法评价其诱导分化效果。结果:Nestin和Vimentin免疫荧光染色阳性的MSCs主要存在于鼻黏膜的固有层内,通过体外培养、传代,可扩增出大量的MSCs,该细胞表达干细胞标志蛋白Nestin,CD133,Vimentin和Sall4;用成骨诱导剂诱导培养后,该细胞的碱性磷酸酶活性明显增强,茜素红染色显示细胞表面形成大量的钙结节;经神经诱导培养液诱导培养后,细胞生长出细长突起并互相连接成网,细胞高表达神经细胞标志蛋白NF-200和BM88。结论:成人鼻腔呼吸黏膜固有层内广泛存在MSCs,该细胞具有多向分化潜能,可作为种子细胞用于自体移植修复骨和神经组织损伤。     

Gene expression changes during step-wise differentiation of embryonic stem cells along the inner ear hair cell pathway

CONCLUSION: Our study outlines an alternative approach for the selection and investigation of genes involved in inner ear function. OBJECTIVE: To gain understanding of the gene pathways involved in the development of the normal cochlea. MATERIALS AND METHODS: Microarray technology currently offers the most efficient approach to investigate gene expression and identify pathways involved in cell differentiation. Epidermal growth factor (EGF) induces cultures derived from the organ of Corti to proliferate and produce new hair cells. Since pluripotent embryonic stem (ES) cells have the capacity to generate all tissues, we induced murine ES cells to differentiate towards ectodermal and neuroectodermal cell types and from there investigated their commitment towards the hair cell lineage in the presence of EGF. Cells were collected at three points along the differentiation pathway and their expression profiles were determined using the Soares NMIE mouse inner ear cDNA library printed in microarray format. RESULTS: Three genes up-regulated after addition of EGF (serine (or cysteine) proteinase inhibitor, clade H, member 1 (Serpinh1), solute carrier family 2 (facilitated glucose transporter), member 10 (Slc2a10) and secreted acidic cysteine-rich glycoprotein (Sparc)) were selected for further analysis and characterization. Of the three genes, Serpinh1 and Slc2a10 have never been implicated in the hearing process.