Morphological differentiation of tau–green fluorescent protein embryonic stem cells into neurons after co-culture with auditory brain stem slices

Most types of congenital and acquired hearing loss are caused by loss of sensory hair cells in the inner ear and their respective afferent neurons. Replacement of spiral ganglion neurons (SGN) would therefore be one prioritized step in an attempt to restore sensory neuronal hearing loss. To initiate an SGN repair paradigm we previously transplanted embryonic neuronal tissue and stem cells (SC) into the inner ear in vivo. The results illustrated good survival of the implant. One such repair, however, would not have any clinical significance unless central connections from the implanted SGN could be established. For the purpose of evaluating the effects of cell transplantation on cochlear nucleus (CN) neurons we have established organotypic brain stem (BS) cultures containing the CN. At present we have used in vitro techniques to study the survival and differentiation of tau–green fluorescent protein (GFP) mouse embryonic stem cells (MESC) as a mono- or co-culture with BS slices. For the co-culture, 300 μm thick auditory BS slices encompassing the CN were prepared from postnatal Sprague–Dawley rats. The slices were propagated using the membrane interface method and the CN neurons labeled with DiI. After 5±2 days in culture a tau-GFP MESC suspension was deposited next to CN in the BS slice. Following deposition the MESC migrated towards the CN. One and two weeks after transplantation the co-cultures were fixed and immunostained with antibodies raised against neuroprogenitor, neuronal, glial and synaptic vesicle protein markers. Our experiments with the tau-GFP MESC and auditory BS co-cultures show a significant MESC survival but also differentiation into neuronal cells. The findings illustrate the significance of SC and auditory BS co-cultures regarding survival, migration, neuronal differentiation and connections.     

Concentration-dependent effect of nerve growth factor on cell fate determination of neural progenitors

Stem cell-based spiral ganglion neuron (SGN) replacement therapy has been proposed to be a promising strategy to restore hearing either via replacing degenerated neurons or by improving the efficacy of cochlear implants which rely on functional neurons. However, lack of suitable donor cells and low survival rate of implanted cells are the major obstacles to successful implementation of therapeutic transplantation. The present study investigated the potential of mouse inner ear statoacoustic ganglion (SAG)-derived neural progenitors (NPs) to differentiate toward SGN-like glutamatergic cells and the influence to cell survival and differentiation when nerve growth factor (NGF) was supplied. We found that SAG-NPs could form neurospheres, proliferate, and differentiate into cells expressing neuronal protein neurofilament and β-III tubulin. NGF affected the cell fate of SAG-NP in a concentration-dependent manner in vitro. Low concentration of NGF (2-5 ng/mL) promoted cell proliferation. Medium concentration of NGF (20-40 ng/mL) stimulated cells to differentiate into bi-polar SGN-like cells expressing glutamatergic proteins. High concentration of NGF (100 ng/mL) could rescue cells from induced apoptosis. In the in vivo study, NGF (100 ng/mL) dramatically enhanced SAG-NP survival rate after implantation into adult mammalian inner ear. This finding raises the possibility to further induce these NPs to differentiate into SGN-like neurons in future in vivo study. In conclusion, given the capability of proliferation and differentiation into SGN-like cells with the supplement of NGF in vitro, SAG-NPs can serve as donor cells in stem cell-based SGN replacement therapy. NGF improved the survival of SAG-NPs not only in vitro but also in vivo.     

人胚胎纹状体区神经干细胞体外生物学特性

目的探讨人胚胎纹状体区神经干细胞在体外的生物学特性。方法从16～20周人胚胎纹状体区分离培养神经干细胞，在体外进行传代、分化，应用免疫组织化学荧光染色等方法，对此区神经干细胞在体外增殖和分化等情况进行研究。结果纹状体区神经干细胞在体外原代培养扩增1月内分裂增殖速度快，在含有EGF和bFGF的培养基中生长最为良好，平均倍增时间为3～4d。分化培养后可以形成各种神经细胞，在原代培养后2周分化情况好，分化为神经元比例高，约占50％左右，在原代培养8周以后分化为神经元的比例下降，约占20％左右。结论人胚胎纹状体区的神经干细胞在体外有较强的自我更新和增殖能力，并表达了干细胞的原始特征，在体外传代过程中，增殖速度随着时间不断下降，其分化为各种神经细胞的能力也不同。培养基中的有丝分裂原对于神经干细胞的增殖和分裂的影响不同。神经球并非均质的，内部仅有部分细胞在分裂增殖。     

海人酸对神经干细胞增殖及分化的影响

背景：神经干细胞对脑组织的修复作用非常有限,约80%新增殖的内源性神经干细胞在6周内死亡,仅0.2%的细胞继续增殖、分化,参与修复。 目的：分析不同剂量海人酸在对神经干细胞增殖及分化的影响。 方法：体外分离并培养新生Wistar大鼠神经干细胞,将神经干细胞分为空白对照组和加入不同浓度梯度的海人酸组,通过免疫组化法和免疫荧光法进行鉴定,MTT比色法测定海人酸对神经干细胞分化的影响,计算分化后神经元和星形胶质细胞比例。 结果与结论：海人酸组贴壁的神经球分化速度较空白对照组快,在同一时间点进行观察,神经细胞的迁移距离较未处理组远。分化5 d后,海人酸组所分化的细胞中,星状细胞较空白对照组多,而神经元样细胞相对较少,培养的细胞具有自我更新和向神经元﹑少突胶质细胞和星形胶质细胞分化的潜能。兴奋性氨基酸海人酸可使部分神经干细胞死亡,但可促进幸存的神经干细胞增殖及分化,并诱导其向星形胶质细胞分化。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

新生大鼠海马源性神经干细胞的分离培养及其向胆碱能神经元定向诱导分化研究(英文)

本研究目的是从新生SD大鼠海马分离、培养神经干细胞并诱导其向胆碱能神经元方向分化。利用含b FGF(20ng/ml)和B27的无血清DMEM/F12培养基培养新生SD大鼠海马分离的具有自我更新和多向分化能力的细胞群,用免疫细胞化学技术检测巢蛋白(nestin),并于分化后分别检查特异性成熟神经细胞、星形胶质细胞、少突胶质细胞的标记抗原β微管蛋白(Tuj1 )、胶质纤维酸性蛋白(GFAP)和半乳糖脑苷脂(Galc)的表达;用鸡胚骨骼肌提取液,诱导神经干细胞向胆碱能神经元方向分化。结果显示:从海马分离的细胞群具有自我更新能力,表达nestin,分化成熟后的细胞表达神经元、星形胶质细胞和少突胶质细胞的特异性抗原;与对照组3. 9%相比,鸡胚骨骼肌提取液可以诱导这些细胞中的9. 6%分化成为胆碱能神经元。提示分离的细胞具有自我更新能力和多向分化潜能,是中枢神经系统的干细胞;在加有鸡胚骨骼肌提取液的培养基诱导下,能向胆碱能神经元方向分化。     

人胚脑神经干细胞的分离培养、克隆和分化

探讨从人胚脑分离培养的神经干细胞在增殖和分化方面的生物学特点。用无血清培养技术从 4月龄人胎中脑组织中分离培养出神经干细胞 ,用有限稀释法获得单细胞克隆球 ,消化后用含 Brd U的培养液培养 ,待形成神经干细胞球后 ,进行 Brd U和nestin免疫荧光检测。取第 4代神经干细胞球用含 10 % FBS的培养液诱导分化 ,3周后分别进行神经元、星形胶质细胞和少突胶质细胞特异性标记物 MAP-2、GFAP和 CNP免疫荧光检测。结果显示 ,单细胞悬液培养 2周后可形成神经干细胞球 ;神经球 Br-d U和 nestin免疫荧光检测均呈阳性 ;神经干细胞分化后呈 MAP-2、GFAP和 CNP阳性的三种类型细胞 ,但分化的神经元数量较少、胞体较小、突起较少。提示从人胚中脑组织中分离得到的神经干细胞具有增殖和自我更新能力 ,并具有分化为神经元、星形胶质细胞和少突胶质细胞的多分化潜能 ;与啮齿类动物相比 ,人神经干细胞增殖速度较慢 ,分化的神经元较少且稍欠成熟     

Neural stem cells differentiation ability of human umbilical cord mesenchymal stromal cells is not altered by cryopreservation

Human umbilical mesenchymal stem cells (HUMSCs) have potential therapeutic use in the recovery of central nervous system injury for their ability to differentiate into neural stem cells. However, for transformed HUMSCs to be constantly available for use during surgery a reliable method of cell storage is necessary. The present study aimed to determine whether a simple method of cryopreservation by slow cooling with Me(2)SO had an effect on the proliferation, secretion and differentiation capacities of HUMSCs. These results demonstrate that cryopreservation has no effect on the phenotype, cell cycle, cell proliferation and the ability to secret neurotrophins. Non-cryopreserved and cryopreserved HUMSCs showed the similar ability to differentiate into neural stem-like cells. There results show that cryopreservation by slow cooling with Me(2)SO is effective to retain the proliferation and neural differentiation ability of HUMSCs, cryopreserved HUMSCs maybe very useful for future clinical applications in neural regenerative medicine.     

大鼠脊髓神经干细胞的鉴定及光镜和电镜观察

本研究应用无血清培养技术对分离的大鼠胚胎脊髓神经干细胞进行体外培养,在倒置显微镜和电镜下观察细胞形态,应用BrdU标记、免疫荧光染色检测细胞增殖、分化情况。免疫荧光显示nestin、MAP2、GFAP以及BrdU/nestin、BrdU/MAP2、Br-dU/GFAP均有阳性表达,说明从大鼠胚胎脊髓可成功分离出神经干细胞,它们分化后可以表达神经元、星形胶质细胞的特异性抗原。脊髓神经干细胞具有自我更新能力,能分化为神经元和星型胶质细胞。     

Cell replacement therapy in the inner ear

The mammalian inner ear is vulnerable to genetic disorders and aging, as well as to injuries caused by overstimulation, ototoxic drugs, and viral infections. Due to the poor regeneration of the sensory epithelium and the spiral ganglion neurons in the adult mammalian inner ear, cell replacement therapy strategies have been proposed to compensate for degeneration and loss of sensory and neuronal cells. Transplantation of stem cells and embryonic neurons into the inner ear has revealed that exogenous cells can survive, migrate, differentiate, and extend neuritic projections in the auditory system of adult mammals. These results suggest that cell replacement therapy could provide an effective future treatment alternative for hearing loss and other inner ear disorders.     

The survival of neural precursor cell grafts is influenced by in vitro expansion

Embryonic neural precursor cells (ENPs) provide a potential alternative for transplantation in neurodegenerative diseases, as they can be expanded in culture, avoiding many of the practical obstacles that limit the application of transplanting primary neurones. However, grafts of ENPs into animal models show variable survival and limited differentiation into neurones. The effect of expansion time on their ability to survive and differentiate may be an important factor in this and has not been examined directly. In these experiments, murine and human ENPs were expanded for short (4 weeks) and long (20 weeks) periods before transplantation into the adult rat striatum. Whereas grafts of both short- and long-term expanded human ENPs survived for 4 weeks following transplantation, by 20 weeks all long-term expanded grafts had disappeared. Murine ENPs behaved similarly: only grafts of short-term expanded ENPs survived at 12 weeks following transplantation. RT-PCR analysis of ENP cultures after 4 and 20 weeks of expansion demonstrated changes in expression of a number of different groups of genes. We conclude that long-term expansion of ENPs profoundly impairs their ability to survive long-term after transplantation into the adult brain. This has implications for the potential use of these cells for neural transplantation strategies.     

Human umbilical cord blood-derived mesenchymal stem cells do not differentiate into neural cell types or integrate into the retina after intravitreal grafting in neonatal rats

This study investigated the ability of mesenchymal stem cells (MSCs) derived from full-term human umbilical cord blood to survive, integrate and differentiate after intravitreal grafting to the degenerating neonatal rat retina following intracranial optic tract lesion. MSCs survived for 1 week in the absence of immunosuppression. When host animals were treated with cyclosporin A and dexamethasone to suppress inflammatory and immune responses, donor cells survived for at least 3 weeks, and were able to spread and cover the entire vitreal surface of the host retina. However, MSCs did not significantly integrate into or migrate through the retina. They also maintained their human antigenicity, and no indication of neural differentiation was observed in retinas where retinal ganglion cells either underwent severe degeneration or were lost. These results have provided the first in vivo evidence that MSCs derived from human umbilical cord blood can survive for a significant period of time when the host rat response is suppressed even for a short period. These results, together with the observation of a lack of neuronal differentiation and integration of MSCs after intravitreal grafting, has raised an important question as to the potential use of MSCs for neural repair through the replacement of lost neurons in the mammalian retina and central nervous system.     

大鼠骨髓间充质干细胞分化为神经干细胞

为了观察骨髓间充质干细胞(BMSCs)分化为神经干细胞(NSCs)的能力,本研究通过贴壁法培养大鼠BMSCs,体外培养扩增纯化后,在细胞传代时用含有表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)、N2、B27的DMEM/F12的培养液制成细胞悬液,并进行诱导,观察诱导后细胞的形态及生长情况,用免疫荧光检测形成的细胞球的巢蛋白(nestin)的表达情况;形成的细胞球在含10%血清的培养液中进一步分化。结果显示:BMSCs在含EGF、bFGF、N2、B27的培养液中,逐渐形成nestin表达阳性的细胞球,在含血清的培养液中能分化为神经元样细胞、星形胶质样细胞及少突胶质样细胞。本研究结果提示经纯化的BMSCs能分化为NSCs,并具有进一步分化的能力。     

神经干细胞在海人酸毁损大鼠海马中的迁移和分化(英文)

本研究旨在观察胎鼠神经干细胞移植入海人酸毁损成年大鼠海马中的迁移和分化情况。立体定位注射海人酸毁损大鼠海马CA1区锥体细胞,毁损一周后,将Hoechst33342标记的神经干细胞移植毁损区,分别于术后1、2、4、8周取材,利用荧光技术和免疫组织化学方法,追踪移植的神经干细胞在毁损侧海马中的存活、迁移和分化情况。结果显示,移植的神经干细胞在海马锥体层呈链状迁移,并分化为MAP2阳性细胞和GFAP阳性细胞。这些结果提示移植的神经干细胞在海马锥体层呈链状迁移,大部分分化为胶质细胞,部分分化为神经元。     

诱导多功能干细胞在视网膜疾病中的应用

背景：视网膜疾病以视网膜细胞损伤或凋亡为特点,缺乏理想的治疗方法。诱导多功能干细胞具有自我更新和多向分化潜能,不存在伦理道德问题,为干细胞移植治疗提供了新的来源,是当前研究的热点。 目的：综述诱导多功能干细胞在视网膜疾病治疗中应用的研究进展。 方法：第一作者应用计算机检索PubMed 相关文献,检索时间为2006年至2011年,检索词“induced pluripotent stem cells,retinal,photoreceptor,retinal pigment epithelium,retinal ganglion cell”并限定文献语言种类为English。共纳入42篇文章进行综述。 结果与结论：诱导多功能干细胞可以诱导分化为视网膜细胞,为细胞移植治疗提供来源,也可以作为疾病的模型来研究发病机制或者药物筛选,因此诱导多功能干细胞治疗视网膜疾病具有很大前景。然而,体细胞成功诱导为诱导多功能干细胞的效率低,并且存在致瘤的风险,有待更进一步研究。     

c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation

The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. Here, we provide genetic evidence for an unexpected function of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors, suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term HSCs. Collectively, our data indicate that c-Myc controls the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSCs and their niche.     

Neuronal cell adhesion molecule (NrCAM) is expressed by sensory cells in the cochlea and is necessary for proper cochlear innervation and sensory domain patterning during development

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