兔胚胎神经干细胞的分离、培养和鉴别

目的：研究兔胎脑神经干细胞体外生长特性,为探讨神经干细胞的临床应用及神经系统的发育奠定基础。方法：采用含碱性成纤维细胞生长因子（bFGF）和表皮细胞生长因子（EGF）的N2无血清培养技术,取18天龄兔胚胎脑组织,分离神经干细胞,并观察分离的细胞体外培养、增殖、分化潜能,免疫组化鉴定。结果：从18天龄兔胎脑皮质和纹状体中成功分离出具有自我更新和多分化潜能的神经干细胞,在无血清培养时细胞呈半贴壁状态生长,形成神经球,可传代。细胞呈Nestin免疫反应阳性;在含血清培养基中培养时则分化,分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原。结论：来自兔胎脑神经干细胞能在体外培养、增殖并保持传代能力。无血清N2EGF、bFGF培养基有利于兔胎脑神经干细胞的存活和增殖,含血清培养基能诱导兔胎脑神经干细胞分化。     

人胚不同脑区神经前体细胞的分离培养及分化特性的比较

目的研究人胚不同脑区神经前体细胞(neural progenitor cells,NPCs)培养及增殖分化特性。方法取14-17周人胚脑区组织,分为新皮质、纹状体、间脑、中脑、后脑和延髓组,悬浮培养。鉴定细胞球巢蛋白抗原的表达,分化及自我更新能力。观察各脑区培养细胞的生长、增殖状况。新皮质、纹状体及间脑来源的神经球分化后,运用免疫荧光细胞化学法比较神经元及星形胶质细胞的比例。结果各脑区培养出的悬浮细胞球巢蛋白抗原阳性,可分化为MAP2或GFAP阳性细胞,且BrdU掺入实验阳性。体外培养第3d,纹状体及间脑组均可见大量神经球,且纹状体组明显多于间脑组;新皮质组传代后可见较多神经球;其它组仅见个别神经球。新皮质、纹状体、间脑来源的NPCs诱导分化后,MAP2或GFAP阳性细胞率各组间比较差异无显著性。结论人胚不同脑区均可培养出NPCs,从易到难依次为纹状体、间脑、新皮质及其它脑区。新皮质、纹状体、间脑来源的NPCs体外分化比例一致。     

人胚与鼠胚神经干细胞体外培养的差异

为比较人胚与鼠胚神经干细胞体外培养的差异。实验采用具有丝裂原作用的细胞生长因子。结合无血清细胞培养技术从人胚和鼠胚皮层分离神经干细胞。在连续传代过程中观察其体外培养特性,免疫荧光染色检测Nestin抗原和分化后特异性成熟神经细胞抗原的表达,并用流式细胞仪检测神经干细胞分化情况。结果表明：（1）使用单一生长因子即可从鼠胚皮层分离神经干细胞,但在人胚却需同时使用多种生长因子,协同使用bFGF,EGF和LIF是人胚神经干细胞体外培养的较佳条件;（2）鼠胚皮层神经干细胞在连续传代过程中增殖速度快于人胚,其Nestin阳性率和BrdU标记的阳性率亦高于人胚,表明其增殖能力明显高于人胚,（3）人胚神经干细胞较鼠胚更易分化为神经元。     

维甲酸和EGF对大鼠脑胚胎神经干细胞增殖和分化的影响

目的 观察全反式维甲酸(RA)和表皮生长因子(EGF)对大鼠胚胎神经干细胞增殖和分化的影响。方法 从大鼠胚胎脑中分离神经干细胞,经RA和EGF处理后,用台盼蓝确定细胞数量,BrdU标记分析细胞生长能力,采用免疫细胞化学法鉴定神经干细胞和分化的神经细胞。结果 20ng／ml EGF和1μmol／LRA处理的培养细胞均显示增殖效应,但EGF处理组增殖速度明显高于RA组,悬浮细胞中有大量nestin和BrdU阳性细胞。用EGF和EGE／RA诱导的神经元分化率分别为17％和31％,而RA处理的神经元分化率显升高至89％。由EGF、EGF／RA和RA诱导的星形胶质细胞分化率分别为83％、69％和11％。结论 EGF主要促进神经干细胞增殖并主要诱导星形胶质细胞的生成,RA主要诱导神经干细胞向神经元分化,二无明显协同效应。     

诱导小鼠胚胎干细胞在体外分化为骨骼肌细胞的实验研究

小鼠胚胎干细胞是从胚泡未分化的内部细胞团中得到的干细胞,它在体外培养的环境中具有无限增殖、自我更新以及多向分化的特性。将小鼠胚胎干细胞在体外诱导分化为肌肉细胞,并且利用这些分化得来的肌肉细胞治疗肌肉退行性疾病,是干细胞研究领域的热点。该实验的目的在于筛选小鼠胚胎干细胞向骨骼肌细胞定向分化的实验条件,有效地将体外单层贴壁培养的小鼠胚胎干细胞诱导分化成骨骼肌细胞。最终发现,10-8mol/L维甲酸(retinoid acid,RA)+0.5%二甲基亚砜(dimethyl sulfoxide,DMSO)组诱导小鼠胚胎干细胞在体外分化成骨骼肌前体细胞的效率最高,分化得到的骨骼肌前体细胞经进一步纯化,能分化为多核的肌管。该实验为治疗肌肉退行性疾病提供了细胞来源,也为研究小鼠胚胎干细胞分化为骨骼肌细胞的机制提供了有利的条件。     

神经干细胞来源的神经元的诱导分裂

为探讨神经干细胞分化成熟的神经元是否能够分裂。实验取材于成年哺乳动物,将神经干细胞体外培养8d后,诱导分化为神经元,然后进一步诱导其分裂。采用连续摄影与NF-160免疫细胞化学方法检测神经元的分裂过程,同时运用PCNA NF-160(或Chat、GABA、GAD)的免疫双标记证明分裂神经元是否为成熟神经元。将神经干细胞体外诱导分化培养8d,直至分化神经元外形成熟,进而加入EGF与bFGF诱导分裂。诱导分裂2d后,观察到有神经元样细胞分裂;同一区域内神经元样细胞的数量不断增加,表现为NF-160阳性。连续拍摄了神经元样细胞的分裂过程,分裂完成后的细胞同样表现为NF-160抗体反应阳性。PCNA NF-160(或Chat、GABA、GAD)的免疫双标记结果显示,一些细胞的胞浆显示为棕色的同时细胞核显示为黑色。结果提示,在一定的条件下,先前所认为的终末分化神经元可以重新进入细胞周期,成熟神经元仍然可以进行分裂增殖和自我更新。     

人胚胎干细胞定向分化为神经前体细胞

采用单层贴壁分化的方法在无血清条件下诱导同源饲养层培养的人胚胎干细胞定向分化，得到了高比例的神经前体细胞（97.5±0.83)%（P＜0.05)。这些神经前体细胞具有分化为神经元、星形胶质细胞和少突胶质细胞的能力。在长期的传代培养中发现，随着培养时间的延长，nestin阳性的神经前体细胞比例下降，同时发育能力也发生了变化。在传代培养的早期，神经前体细胞发育为神经元的比例很高，几乎没有胶质细胞分化出来。随着培养时间的延长，胶质细胞的比例逐渐上升。这与体内神经系统的发育过程非常相似。进一步研究发现具有bHLH (basic helix-loop-helix) 结构域的转录因子neurogenein2(Ngn2) 和Olig2可能在这一变化中起重要作用。因此，人胚胎干细胞来源的神经前体细胞能够模拟体内神经发育的模式，为在体外研究人的神经发育和再生医学奠定了基础。     

从人胚胎干细胞畸胎瘤中有效获取间充质干细胞和神经前体细胞

通过人胚胎干细胞(human embryonic stem cells,hESC)体外分化方法和畸胎瘤形成可以分化获得多种成体细胞．但目前尚不清楚是否可以从hESCs畸胎瘤中分离某些特异性细胞．通过体外筛选方法,有效地从hESCs畸胎瘤中分离出神经前体细胞(neural progenitor cells,NPCs)和间充质干细胞(mesenchymal stem cells,MSCs)．这种hESCs畸胎瘤来源的NPCs和MSCs与体内神经前体细胞和间充质干细胞有着相似的分子标记和特性,并具有进一步的分化潜能——分别可以诱导成为神经元、神经胶质细胞、脂肪细胞和骨骼细胞等．根据人胚胎干细胞畸胎瘤中含有不同分化阶段的外胚层、中胚层和内胚层的组织或细胞,认为人胚胎干细胞畸胎瘤可以作为另一个细胞来源以获取多种(包括人胚胎干细胞体外分化难以得到的)各种前体/干细胞和终末分化细胞．     

间充质干细胞长期传代培养条件的研究

间充质干细胞(mesenchymal stem cells,MSCs)是存在于成体组织间质部分的多能前体细胞,在体外具有自我更新增殖及向成脂、成骨、成软骨分化的潜能,在组织工程和细胞治疗方面具有广阔的应用前景。MSCs在体外长期培养获得足够数量的细胞是MSCs应用的一个重要因素。然而,目前还没有建立MSCs长期传代培养的最适培养体系。该文分别从培养体系中的基础培养基、血清和生长因子对于MSCs细胞长期传代培养的影响进行了论述,旨在为建立MSCs体外长期传代生长的最适培养体系提供理论依据。     

人神经干细胞的体外生物学特性

本实验利用有丝分裂因子,体外诱导生成人神 经干细胞(NSCs),观察其生长特性并进行鉴定。取胎龄10-22周的大脑半球,分散细胞后种于添加表皮生长因子(EGF,20ng/ml)和/或碱性成纤维生长因子(bFGF,20ng/ml)的培养基中。利用免疫组织化学方法鉴定分化后的细胞类型。同时,进行细胞克隆分析、传代培养及端粒酶活性检测。结果显示:NSCs呈悬浮生长的干细胞球,其特异性抗原nestin阳性。NSCs具有增殖能力,可连续传代而不丢失其增殖和多分化潜能的干细胞特性。撤除EGF和bFGF的作用,细胞停止分裂,并分化为神经元、星形胶质细胞和少突胶质细胞。克隆分析显示NSCs生长呈密度依赖性。人NSCs表达较低的端粒酶水平,并随培养时间延长而下调。研究表明,利用有丝分裂因子,可在体外成功诱导生成人NSCs,其生长,分化受内外源因素的调节,相关的机制还有待阐明。     

A gradient of responsiveness to the growth-promoting activity of ZPA (zone of polarizing activity) in the chick limb bud

Limb bud mesoderm of stage 22-23 embryos was dissected into four pieces along the anteroposterior axis and dissociated cells of each region were separately cultured under various conditions. When the cells were cultured in medium containing 0.1% fetal calf serum (serum-poor medium) only a slight increase in cell number occurred in the cultures of all four regions. However, when the cells were cultured in medium containing 10% FCS, only cells of two central regions proliferated rapidly, and no growth promotion was observed in cells in the most anterior and posterior regions. Using the serum-poor medium, we examined the growth-promoting effects of cocultured limb bud fragments and of some growth factors on the cells of four regions. Anterior, distal, and proximal fragments promoted cell proliferation and their promotive effect on the cells of each region was equal. On the other hand, posterior fragments (containing ZPA) showed stronger promotive effects on preaxial cells than on postaxial cells. For comparison with the growth-promotive effect of the posterior fragment, fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin, and retinoic acid were tested in cell culture. FGF showed position-dependent growth promotion, while EGF and insulin promoted growth in the cells of all four regions to a similar degree. Retinoic acid showed no effect on cell growth at low concentrations, and was rather toxic at high concentrations. These results suggest that the cells of the posterior region secrete an FGF-like growth factor(s), which controls normal limb development and experimental duplicate formation.     

Effect of epidermal growth factor on cultured adult rat hepatocytes

When adult rat hepatocytes were cultured in plastic Petri dishes in a medium containing insulin and glucagon, supplementation with epidermal growth factor (EGF) had a pronounced effect on their viability, morphology, and biochemical integrity. Transmission and scanning electron microscopic studies showed that after 1 week cells denied EGF accumulated numerous non-electron-dense bodies and filamentous whorls, had irregular nuclei, and exhibited atypical cell surfaces. In contrast, cells grown for 2-3 weeks in the presence of EGF had well-preserved cellular organelles and remained as an epithelial-like monolayer. After 3 weeks EGF-exposed cultures were still inducible for liver-specific tyrosine aminotransferase, and both rat albumin and rat transferrin were recoverable from the culture medium. Virtually no viable cells were present at 3 weeks in EGF-deprived cultures.     

脐带血造血干/祖细胞体外分化为不同阶段红系祖细胞的动力学观察

目的:探讨体外培养脐带血单个核细胞定向诱导分化为不同阶段红系祖细胞的动力学变化情况。方法:用0.5%甲基纤维素沉降脐带血红细胞及人淋巴细胞分离液密度梯度离心法得到单个核细胞,在含EPO、SCF、IGF-1等细胞因子的无血清培养体系中诱导其定向分化为红系祖细胞,观察细胞增殖、存活率、细胞集落形成情况,并检测不同阶段细胞红系特异性表面标志CD71和CD235a的表达。结果:随着培养时间的延长,细胞数逐渐增多,14 d细胞可扩增140倍左右,收集诱导后的细胞进行瑞氏吉姆萨染色,可见大量红系祖细胞,诱导后的细胞集落形成能力强,形成的克隆大部分为红系集落。诱导过程中,14 d前CD71、CD235a的表达逐渐增高。按细胞表面标志表达的不同可将诱导的细胞分为4群,分别对应红系祖细胞的不同阶段;随着诱导天数的增加,各时间点细胞对应的早期红系祖细胞群(P2、P3)比例逐渐下降,中晚期红系祖细胞群(P4、P5)的比例逐渐上升。结论:无血清培养基添加细胞因子组合的红系诱导培养体系可较好地诱导扩增红系祖细胞,流式分选可获得相对均一而处于不同分化阶段的红系祖细胞群体。获得了红系祖细胞体外分化的动力学数据,为今后进一步优化红系诱导分化体系获得均一的红系祖细胞奠定了基础,并对未来利用干细胞制备均一的红系祖细胞应用于临床治疗有一定的指导作用。     

Characterisation of stroma-dependent blast colony-forming cells in human marrow

Human bone marrow contains a population of haemopoietic progenitor cells that can be distinguished by their ability to adhere to preformed stromal layers (cultured in the presence of methylprednisolone [MP+] and form blast cell colonies. The stromal layers function in the colony assay after they have been heavily irradiated but not after they have been passaged. The binding of the progenitor cells to the stromal cells is complete after 2 hours of coincubation, and stromal layers of 9.6 cm2 can provide adhesion sites for at least 2,000 blast colony-forming cells. The blast colony-forming cells were shown by micromanipulation to self-renew as well as to give rise to multipotential and lineage-committed colony-forming progenitor cells.     

Epidermal growth factor regulates the development of stem and progenitor Leydig cells in rats

Epidermal growth factor (EGF) has many physiological roles. However, its effects on stem and progenitor Leydig cell development remain unclear. Rat stem and progenitor Leydig cells were cultured with different concentrations of EGF alone or in combination with EGF antagonist, erlotinib or cetuximab. EGF (1 and 10 ng/mL) stimulated the proliferation of stem Leydig cells on the surface of seminiferous tubules and isolated CD90⁺ stem Leydig cells and progenitor Leydig cells but it blocked their differentiation. EGF also exerted anti‐apoptotic effects of progenitor Leydig cells. Erlotinib and cetuximab are able to reverse EGF‐mediated action. Gene microarray and qPCR of EGF‐treated progenitor Leydig cells revealed that the down‐regulation of steroidogenesis‐related proteins (Star and Hsd3b1) and antioxidative genes. It was found that EGF acted as a proliferative agent via increasing phosphorylation of AKT1. In conclusion, EGF stimulates the proliferation of rat stem and progenitor Leydig cells but blocks their differentiation.     

Isolation, culture and identification of epidermal stem cells from newborn mouse skin

In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. Epidermal stem cells represent a promising source of stem cells, and their culture has great potential in scientific research and clinical application. However, no single method has been universally adopted for identifying and isolating epidermal stem cells. Here, we reported the isolation and characterization of putative epidermal stem cells from newborn mouse skin. The keratinocytes were separated enzymatically. Putative epidermal stem cells were selected by rapid adherence on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded after 10 min, and the attached cells were cultured in a defined culture medium. The isolated cells showed the typical epidermal stem cell morphology. Immunofluorescence indicated that the cells were strongly stained for β₁ integrin family of extracellular matrix receptors. In conclusion, mouse putative epidermal stem cells were successfully isolated from newborn mouse epidermis on the basis of high rapid adhesion to extracellular matrix proteins and cultured in vitro.     

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.To further characterize each tumor''s BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 °C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.     

Clonal proliferation of multipotent stem/progenitor cells in the neonatal and adult salivary glands

Salivary gland stem/progenitor cells are thought to be present in intercalated ductal cells, but the fact is unclear. In this study, we sought to clarify if stem/progenitor cells are present in submandibular glands using colony assay, which is one of the stem cell assay methods. Using a low-density culture of submandibular gland cells of neonatal rats, we developed a novel culture system that promotes single cell colony formation. Average doubling time for the colony-forming cells was 24.7 (SD=+/-7.02)h, indicating high proliferative potency. When epidermal growth factor (EGF) and hepatocyte growth factor (HGF) were added to the medium, the number of clonal colonies increased greater than those cultured without growth factors (13.2+/-4.18 vs. 4.5+/-1.73). The RT-PCR and immunostaining demonstrated expressing acinar, ductal, and myoepithelial cell lineage markers. This study demonstrated the presence of the salivary gland stem/progenitor cells that are highly proliferative and multipotent in salivary glands.