Complex extracellular matrices promote tissue-specific stem cell differentiation

Most cells in tissues contact an extracellular matrix on at least one surface. These complex mixtures of interacting proteins provide structural support and biological signals that regulate cell differentiation and may be important for stem cell differentiation. In this study, we have grown a rhesus monkey embryonic stem cell line in the presence of various extracellular matrix components in monolayer, in a NASA-developed rotating wall vessel bioreactor in vitro, and subcutaneously in vivo. We find that individual components of the extracellular matrix, such as laminin-1 or collagen I, do not influence the growth or morphology of the cells. In contrast, a basement membrane extract, Matrigel, containing multiple extracellular matrix components, induces the cells within 4 days to form immature glandular- and tubular-like structures, many of which contain a lumen with polarized epithelium and microvilli. Such structures were seen in vitro when the cells were grown in the bioreactor and when the cells were injected into mice. These tubular- and glandular-like structures were polarized epithelia based on immunostaining for laminin and cytokeratin. The cell aggregates and tumors also contained additional mixed populations of cells, including mesenchymal cells and neuronal cells, based on immunostaining with vimentin and neuronal markers. An extract of cartilage, containing multiple cartilage matrix components, promoted chondrogenesis in vivo where alcian blue-stained cartilage nodules could be observed. Some of these nodules stained with von Kossa, indicating that they had formed calcified cartilage. We conclude that extracellular matrices can promote the differentiation of embryonic stem cells into differentiated cells and structures that are similar to the tissue from which the matrix is derived. Such preprogramming of cell differentiation with extracellular matrices may be useful in targeting stem cells to repair specific damaged organs.     

影响神经干细胞增殖分化的因素

传统认为哺乳动物和人脑神经细胞一经发育成熟即不再进行增殖、分化 ,不具备再生能力 ,只有细胞的不断退化和死亡 ,因此中枢神经系统损伤后基本上不能恢复。近年来的研究发现 ,胚胎早期脑室和脑室下区、成年脑室区、纹状体、海马齿状回、脊髓等部位神经干细胞分布广泛 ,这些神经干细胞可以分化为神经元、星形胶质细胞和少突胶质细胞。随着对干细胞研究的深入 ,人们已能利用无血清培养、单克隆技术及免疫荧光化学技术对干细胞进行分离、培养和纯化。深入研究脑发育过程中的神经干细胞的增殖分化机制 ,搞清神经干细胞在成年脑内增殖、分化的影…     

Endothelial cells promote neural stem cell proliferation and differentiation associated with VEGF activated Notch and Pten signaling

To investigate whether and how endothelial cells affect neurogenesis, we established a system to co‐culture endothelial cells and brain slices of neonatal rat and observed how subventricular zone cells differentiate in the presence of endothelial cells. In the presence of endothelial cells, neural stem cells increased in number, as did differentiated neurons and glia. The augmentation of neurogenesis was reversed by diminishing vascular endothelial growth factor (VEGF) expression in endothelial cells with RNA interference (RNAi). Microarray analysis indicated that expression levels of 112 genes were significantly altered by co‐culture and that expression of 81 of the 112 genes recovered to normal levels following RNAi of VEGF in endothelial cells. Pathway mapping showed an enrichment of genes in the Notch and Pten pathways. These data indicate that endothelial cells promote neural stem cell proliferation and differentiation associated with VEGF, possibly by activating the Notch and Pten pathways. Developmental Dynamics 239:2345–2353, 2010. © 2010 Wiley‐Liss, Inc.     

3D surface topology guides stem cell adhesion and differentiation

Polymerized high internal phase emulsion (polyHIPE) foams are extremely versatile materials for investigating cell–substrate interactions in vitro. Foam morphologies can be controlled by polymerization conditions to result in either open or closed pore structures with different levels of connectivity, consequently enabling the comparison between 2D and 3D matrices using the same substrate with identical surface chemistry conditions. Additionally, here we achieve the control of pore surface topology (i.e. how different ligands are clustered together) using amphiphilic block copolymers as emulsion stabilizers. We demonstrate that adhesion of human mesenchymal progenitor (hES-MP) cells cultured on polyHIPE foams is dependent on foam surface topology and chemistry but is independent of porosity and interconnectivity. We also demonstrate that the interconnectivity, architecture and surface topology of the foams has an effect on the osteogenic differentiation potential of hES-MP cells. Together these data demonstrate that the adhesive heterogeneity of a 3D scaffold could regulate not only mesenchymal stem cell attachment but also cell behavior in the absence of soluble growth factors.     

Electroconductive polymeric nanowire templates facilitates in vitro C17.2 neural stem cell line adhesion, proliferation and differentiation

Stem cells still remain one of the most exciting and lucrative options for treatment of a variety of nervous system disorders and diseases. Although there are neural stem cells present in adults, the ability of both the peripheral and central nervous system for self-repair is limited at best. As such, there is a great need for a tissue engineering approach to solve nervous system disorders and diseases. In this study, we have developed electrically conductive surfaces with controlled arrays of high aspect ratio nanowires for the growth and maintenance of neural stem cells. The nanowire surfaces were fabricated from polycaprolactone using a novel nanotemplating technique, and were coated with an electrically conductive polymer, polypyrrole. The polypyrrole-coated nanowire surfaces were characterized using scanning electron microscopy and X-ray photoelectron spectroscopy. Additionally, the surface resistance of polypyrrole-coated nanowire surfaces was measured. C17.2 neural stem cells were used to evaluate the efficacy of the polypyrrole-coated nanowire surfaces to promote cell adhesion, proliferation and differentiation. The results presented here indicate significantly higher cellular adhesion and proliferation on polypyrrole-coated nanowire surfaces as compared to control surfaces. The differentiation potential of polypyrrole nanowire surfaces was also evaluated by immunostaining key neuronal markers that are expressed when NSCs differentiate into their respective neural lineages.     

神经干细胞分化调控机制的研究进展

神经干细胞（NSCs）是一类具有自我更新和多方向分化潜能的干细胞,可以分化为神经元,星形胶质细胞和少突胶质细胞。自从1992年Reynolds等从小鼠纹状体中分离到神经干细胞之后,相关的研究已经取得了很大的进展。然而由于中枢神经系统内神经干细胞数量较少,而神经系统损伤后移植的外源的神经干细胞大多分化为神经胶质细胞,进而形成瘢痕组织,限制了神经系统的恢复。因此,如何实现神经干细胞的定向诱导分化成为当前该领域的核心问题。     

骨髓基质细胞促进人胚神经干细胞向神经元的分化

目的:探讨骨髓基质细胞(BMSCs)对人胚神经干细胞(NSCs)分化的影响。方法:采用机械法分离人胚NSCs,成球法进行传代培养,采用免疫荧光染色检测神经上皮干细胞蛋白(Nestin)的表达鉴定NSCs。按培养方式不同,分为NSCs自然分化组、BMSCs和NSCs直接接触共培养组及Transwell共培养组,采用免疫细胞荧光法及免疫印迹法检测各组神经元和星形胶质细胞标志物的表达。结果:在直接接触共培养组和transwell共培养组中,免疫荧光染色显示神经元标志物NSE阳性细胞率明显高于自然分化组,而星形胶质细胞标志物GFAP阳性细胞率低于自然分化组。免疫印迹检测显示Transwell共培养组中NSE表达量显著高于自然分化组,而GFAP表达量低于自然分化组。结论:BMSCs具有促进NSCs向神经元分化的作用。     

Modular peptides promote human mesenchymal stem cell differentiation on biomaterial surfaces

Molecular design strategies in biomedical applications often involve creating modular “fusion” proteins, in which distinct domains within a single molecule can perform multiple functions. We have synthesized a new class of modular peptides that include a biologically active sequence derived from the growth factor BMP-2 and a series of hydroxyapatite-binding sequences inspired by the N-terminal α-helix of osteocalcin. These modular peptides can bind in a sequence-dependent manner to the surface of “bone-like” hydroxyapatite coatings, which are nucleated and grown on a biodegradable polymer surface via a biomimetic process. The BMP-2-derived sequence of the modular peptides is biologically active, as measured by its ability to promote osteogenic differentiation of human mesenchymal stem cells. Our study indicates that the modular peptides described here are multifunctional, and the characteristics of this approach suggest that it can potentially be applied to a range of biomaterials for regenerative medicine applications.     

The correlation between human adipose-derived stem cells differentiation and cell adhesion mechanism

In recent years, research in the areas of stem cells has dramatically increased, including studies of cellular adhesion to a substrate. We sought to determine the adhesive properties of human adipose-derived stem cells (hASCs) for extracellular matrix proteins. The adhesion of hASCs to collagens and laminin was completely inhibited by a monoclonal antibody, Mab 2253, which binds to the β1 integrin subunit. These data indicate that hASC adhesion to collagens and laminin was exclusively mediated by an integrin. Cell adhesion on fibronectin (Fn) was inhibited by the heparin-binding peptide (HBP) in the presence of Mab 2253, but not by either Mab 2253 or HBP alone. These results indicate that both the β1 subunit and the heparan sulfate proteoglycan participated in the cell adhesion to Fn. Microscopic views showed extensive spreading of hASCs cultured on Fn, whereas the cells maintained a round shape when cultured on a heparin-binding domain (HBD) substrate. hASCs differentiated into adipocytes, which stained positive for lipid vacuoles by Oil Red-O analysis, more readily on HBD substrate than on FN substrate. These results suggest that hASCs have an adhesion mechanism for the HBD of Fn and hASC morphology is controlled by the adhesion mechanism and strongly correlated with adipogenic differentiation.     

冬虫夏草含药血清诱导神经干细胞定向分化的影响

目的探讨冬虫夏草中含药血清诱导神经干细胞（NSCs）定向分化的作用。方法取新生24 h内的Wistar大鼠大脑海马区分离培养神经干细胞三代后,加入低、中、高剂量冬虫夏草含药血清与对照血清进行培养,观察其对神经干细胞影响,通过MTT检测神经细胞生长曲线和免疫组化法检测神经干细胞向神经元分化的情况。结果新生大鼠海马分离培养的神经克隆球,经免疫细胞化学染色检测为Nestin阳性细胞。神经干细胞贴壁分化的细胞呈NSE、GFAP阳性。冬虫夏草低、中剂量组诱导神经干细胞分化为神经元的阳性细胞明显高于对照组,且呈量效依赖关系,高剂量组与对照组没有明显差别。结论冬虫夏草含药血清可体外诱导神经干细胞向神经元方向分化,在一定范围内存在量效依赖关系。     

Human embryonic stem cell differentiation toward regional specific neural precursors

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that have the capacity to differentiate into a wide variety of cell types. This potentiality represents a promising source to overcome many human diseases by providing an unlimited supply of all cell types, including cells with neural characteristics. Therefore, this review summarizes early neural development and the potential of hESCs to differentiate under in vitro conditions, examining at the same time the potential use of differentiated hESCs for therapeutic applications for neural tissue and cell regeneration.     

Brain injury activates microglia that induce neural stem cell proliferation ex vivo and promote differentiation of neurosphere-derived cells into neurons and oligodendrocytes

Brain damage, such as ischemic stroke, enhances proliferation of neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ). To date, no reliable in vitro systems, which can be used to unravel the potential mechanisms underlying this lesion-induced effect, have been established. Here, we developed an ex vivo method to investigate how the proliferation of NSPCs changes over time after experimental stroke or excitotoxic striatal lesion in the adult rat brain by studying the effects of microglial cells derived from an injured brain on NSPCs. We isolated NSPCs from the SVZ of brains with lesions and analyzed their growth and differentiation when cultured as neurospheres. We found that NSPCs isolated from the brains 1-2 weeks following injury consistently generated more and larger neurospheres than those harvested from naive brains. We attributed these effects to the presence of microglial cells in NSPC cultures that originated from injured brains. We suggest that the effects are due to released factors because we observed increased proliferation of NSPCs isolated from non-injured brains when they were exposed to conditioned medium from cultures containing microglial cells derived from injured brains. Furthermore, we found that NSPCs derived from injured brains were more likely to differentiate into neurons and oligodendrocytes than astrocytes. Our ex vivo system reliably mimics what is observed in vivo following brain injury. It constitutes a powerful tool that could be used to identify factors that promote NSPC proliferation and differentiation in response to injury-induced activation of microglial cells, by using tools such as proteomics and gene array technology.     

Nanofiber matrices promote the neuronal differentiation of human embryonic stem cell-derived neural precursors in vitro

The potential of human embryonic stem (ES) cells as experimental therapies for neuronal replacement has recently received considerable attention. In view of the organization of the mature nervous system into distinct neural circuits, key challenges of such therapies are the directed differentiation of human ES cell-derived neural precursors (NPs) into specific neuronal types and the directional growth of axons along specified trajectories. In the present study, we cultured human NPs derived from the NIH-approved ES line BGO1 on polycaprolactone fiber matrices of different diameter (i.e., nanofibers and microfibers) and orientation (i.e., aligned and random); fibers were coated with poly-L-ornithine/laminin to mimic the extracellular matrix and support the adhesion, viability, and differentiation of NPs. On aligned fibrous meshes, human NPs adopt polarized cell morphology with processes extending along the axis of the fiber, whereas NPs on plain tissue culture surfaces or random fiber substrates form nonpolarized neurite networks. Under differentiation conditions, human NPs cultured on aligned fibrous substrates show a higher rate of neuronal differentiation than other matrices; 62% and 86% of NPs become TUJ1 (+) early neurons on aligned micro- and nanofibers, respectively, whereas only 32% and 27% of NPs acquire the same fate on random micro- and nanofibers. Metabolic cell activity/viability studies reveal that fiber alignment and diameter also have an effect on NP viability, but only in the presence of mitogens. Our findings demonstrate that fibrous substrates serve as an artificial extracellular matrix and provide a microenviroment that influences key aspects of the neuronal differentiation of ES-derived NPs.     

小鼠视网膜片层化及神经干细胞的增殖与分化

目的 通过观察小鼠视网膜神经干细胞增殖与双极细胞分化过程,研究视网膜的发生及片层化。方法 应用免疫荧光、5’-溴脱氧尿嘧啶核苷（BrdU）检测技术和HE染色法对胚胎及出生后小鼠视网膜形态结构及神经干细胞的增殖、分化进行观察,对视网膜BrdU和蛋白激酶Cα(PKC-α)阳性细胞密度进行统计。结果 1.小鼠视网膜在胚胎时期分化出色素上皮层、神经母细胞层和神经节细胞层。出生后,神经母细胞层逐渐分化出各个层,至小鼠睁眼时基本分化完全。2.小鼠视网膜干细胞在胚胎期大量增殖,出生后增殖放慢并逐渐分化为各类细胞。经统计分析发现,视网膜干细胞在胚胎时期数量逐渐增多,到出生当天数量达到最大值,出生后,神经干细胞开始分化,数量逐渐减少。3.小鼠视网膜双极细胞从出生后第5天（P5）开始发育,至P20时发育完全。结论 小鼠视网膜的片层化与其功能的成熟相一致,视网膜的神经干细胞在出生后前期为分化高峰期,逐渐分化为不同类型的细胞。P10以后仅在睫状体处存在神经干细胞,可能与成年以后的修复功能相关。     

神经干细胞的干性维持机制*

背景：神经干细胞具有强大的自我更新和多向分化潜能,是治疗神经系统疾病的研究热点。 目的：总结神经干细胞的生物学特性及干细胞维持和分化机制的研究进展。 方法：由第一作者检索PubMed数据库1994-01/2011-05及清华同方中文系列数据库2000-01/2011-05涉及神经干细胞的生物学特征及干细胞维持和分化的文献,英文关键词为“Neural stem cells,cell factors,ips,microenvironment”,中文关键词为“神经干细胞,细胞因子,诱导多能干细胞,微环境”,排除陈旧性、重复性文献,保留55篇进行归纳总结。 结果与结论：神经干细胞是神经系统发育过程中保留下来的具有自我更新和多向分化潜能的原始细胞,其免疫原性低、无致瘤性,在神经系统类疾病的治疗方面拥有巨大的应用前景。神经干细胞自我更新与多向分化潜能的维持(干性维持)受其内源因子的调控,这些因子包括Tlx、Sox2、Hes、Bmi-1等转录因子,同时这一干性维持过程也受外源性信号的调控,外源性信号主要是指神经干细胞所处的微环境,包括神经干细胞周围基质细胞、细胞因子等方面。      

The effect of ultra-nanocrystalline diamond films on the proliferation and differentiation of neural stem cells

The interaction of ultra-nanocrystalline diamond (UNCD) with neural stem cells (NSCs) has been studied along with its surface modification in order to improve its function as a biomaterial. Hydrogen- and oxygen-terminated UNCD films were compared with standard grade polystyrene in terms of their impact on the growth, expansion and differentiation of NSCs. When NSCs were cultured on these substrates in low serum and without any differentiating factors, hydrogen-terminated UNCD films spontaneously induced cell proliferation and neuronal differentiation. Oxygen-terminated UNCD films were also shown to further improve neural differentiation, with a preference to differentiate into oligodendrocytes. Hence, controlling the surface properties of UNCD could manipulate the differentiation of NSCs for different biomedical applications. These observations raise the potential for the use of UNCD as a biomaterial for central nervous system transplantation and tissue engineering.     

Knockdown of tissue nonspecific alkaline phosphatase impairs neural stem cell proliferation and differentiation

In the adult mammalian brain the subependymal layer of the lateral ventricles houses neural stem cells giving rise to young neurons migrating towards the olfactory bulb. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, differentiation and functional integration are poorly understood. Neural progenitors in situ express the tissue nonspecific form of alkaline phosphatase (TNAP), a cell surface-located nonspecific phosphomonoesterase capable of hydrolyzing extracellular nucleotides. To gain insight into the functional role of TNAP in cultured multipotent neural stem cells we applied a knockdown protocol using RNA interference with shRNA and retroviral infection. We show that TNAP knockdown reduces cell proliferation and differentiation into neurons or oligodendrocytes. This effect is abrogated by addition of alkaline phosphatase to the culture medium. Our results suggest that TNAP is essential for NSC proliferation and differentiation in vitro and possibly also in vivo.     

Engineering the cell-material interface for controlling stem cell adhesion, migration, and differentiation

The effective utilization of stem cells in regenerative medicine critically relies upon our understanding of the intricate interactions between cells and their extracellular environment. While bulk mechanical and chemical properties of the matrix have been shown to influence various cellular functions, the role of matrix interfacial properties on stem cell behavior is unclear. Here, we report the striking effect of matrix interfacial hydrophobicity on stem cell adhesion, motility, cytoskeletal organization, and differentiation. This is achieved through the development of tunable, synthetic matrices with control over their hydrophobicity without altering the chemical and mechanical properties of the matrix. The observed cellular responses are explained in terms of hydrophobicity-driven conformational changes of the pendant side chains at the interface leading to differential binding of proteins. These results demonstrate that the hydrophobicity of the extracellular matrix could play a considerably larger role in dictating cellular behaviors than previously anticipated. Additionally, these tunable matrices, which introduce a new control feature for regulating various cellular functions offer a platform for studying proliferation and differentiation of stem cells in a controlled manner and would have applications in regenerative medicine.     

Inkjet printing of macromolecules on hydrogels to steer neural stem cell differentiation

Inkjet printing allows for the rapid and inexpensive printing of cells, materials, and protein molecules. However, the combination of inkjet printing and control of neural stem cell (NSC) multipotency and differentiation has remained unexplored. We used an inkjet printer (Canon BJC-2100) to print biologically active macromolecules on poly-acrylamide-based hydrogels (HydroGel(TM)), which were subsequently seeded with primary fetal NSCs. NSCs cultured on areas printed with fibroblast growth factor-2 (FGF2) remained undifferentiated, consistent with the effects of FGF2 when administered in solution. NSCs cultured in parallel on the same hydrogels but in areas printed with ciliary neurotrophic factor (CNTF) or fetal bovine serum (FBS) displayed a rapid induction of markers for astrocytic (glial fibrillary acidic protein, GFAP) or smooth muscle (smooth muscle actin, SMA) differentiation, respectively. These results are consistent with known actions of CNTF and FBS on NSCs. Importantly, NSCs cultured on a printed gradient of increasing levels of CNTF showed a linear increase in numbers of cells expressing GFAP, demonstrating a functional gradient of CNTF. Lastly, genetically modified NSCs proved to respond properly to printed macromolecules, suggesting that inkjet printing can successfully be combined with gene delivery to achieve effective control of stem cell differentiation.