含光诱导片段定向滑动多肽水凝胶诱导胚胎干细胞的外胚层分化（英文）

由细胞内行为或外源力引起的干细胞龛中存在的机械信号对干细胞的自我恢复和分化等基本功能具有重要影响.然而,关于具有分子机械运动产生的内在机械信号的人工细胞外基质鲜有报道.在此,我们报道了含光诱导片段定向滑动的机械动态水凝胶的合成及其作为人工细胞外基质在调节胚胎干细胞(ESC)分化中的功能.通过引入光笼蔽的半胱氨酸残基调控亲疏水交替多肽的自组装制备机械动态水凝胶.光笼蔽多肽组装体在光照射下转化为热力学非平衡的非笼蔽多肽双分子层,其进一步发生热力学有利的疏水性塌陷转变诱导的片段定向滑动.在机械动态水凝胶上培养鼠胚胎干细胞,该片段定向滑动诱导干细胞向外胚层谱系定向分化.进一步揭示了机械动态水凝胶促进机械转导蛋白YAP进入细胞核,表明其用于ESCs定向分化的潜在机械转导机制.细胞定向分化结果表明了机械动态水凝胶作为潜在的生物材料,有望用于疾病治疗和组织再生.     

Shrink-film configurable multiscale wrinkles for functional alignment of human embryonic stem cells and their cardiac derivatives

A biomimetic substrate for cell-culture is fabricated by plasma treatment of a prestressed thermoplastic shrink film to create tunable multiscaled alignment "wrinkles". Using this substrate, the functional alignment of human embryonic stem cell derived cardiomyocytes is demonstrated.     

Systems level approach reveals the correlation of endoderm differentiation of mouse embryonic stem cells with specific microstructural cues of fibrin gels

Stem cells receive numerous cues from their associated substrate that help to govern their behaviour. However, identification of influential substrate characteristics poses difficulties because of their complex nature. In this study, we developed an integrated experimental and systems level modelling approach to investigate and identify specific substrate features influencing differentiation of mouse embryonic stem cells (mESCs) on a model fibrous substrate, fibrin. We synthesized a range of fibrin gels by varying fibrinogen and thrombin concentrations, which led to a range of substrate stiffness and microstructure. mESCs were cultured on each of these gels, and characterization of the differentiated cells revealed a strong influence of substrate modulation on gene expression patterning. To identify specific substrate features influencing differentiation, the substrate microstructure was quantified by image analysis and correlated with stem cell gene expression patterns using a statistical model. Significant correlations were observed between differentiation and microstructure features, specifically fibre alignment. Furthermore, this relationship occurred in a lineage-specific manner towards endoderm. This systems level approach allows for identification of specific substrate features from a complex material which are influential to cellular behaviour. Such analysis may be effective in guiding the design of scaffolds with specific properties for tissue engineering applications.     

表达绿色荧光蛋白基因的花鲈胚胎干细胞株的建立及其体外分化

以带有绿色荧光标记的基因(pCMV-EGFP)为报告基因,用Genejammer、Genejuice和Metafectene三种脂质体介导花鲈胚胎干细胞(LJES1)的基因转移.实验发现,Genejammer介导的细胞转化效率最高,高达27.3%,其余分别为12.1%和5.3%.转移绿色荧光蛋白(GFP)基因的LJES1细胞经过药物筛选和单克隆化培养,获得了表达GFP基因的阳性克隆细胞株,经PCR对GFP阳性细胞株的基因组DNA及提取的RNA扩增,获得了目的条带,证实了GFP基因已经整合到LJES1细胞的基因组中,并获得了正常的表达.通过体外诱导,GFP阳性细胞能够分化为神经细胞、肌肉细胞、成纤维细胞等,用悬滴法培养获得了GFP阳性细胞的拟胚体,证实了经过长期的药物筛选后,LJES1细胞仍然保持着发育的多能性.这一研究,为进一步利用海水鱼类胚胎干细胞进行遗传操作及基因工程的研究提供了方法上的探索.     

Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

Abstract

Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs) direct embryonic stem (ES) cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types.     

High-speed tracking of murine cardiac stem cells by harmonic nanodoublers

Potassium niobate nonlinear nanoparticles are used for the first time to monitor the evolution of embryonic stem cells (ESC) by second harmonic microscopy. These particles feature the complete absence of photo-bleaching and unlimited excitation wavelength flexibility. The potential of this approach is made evident for tissue-regeneration studies and applications, by capturing a high-speed movie of ESC-derived cardiomyocytes autonomously beating within a cluster. Time-resolved data are analyzed to retrieve 3D information of the contraction pattern at the cellular level.     

The characteristics of nuclear membrane fluctuations in stem cells

We analyse the stem cell nucleus shape fluctuation spectrum obtained from optical confocal microscopy on an hour time scale with 10 s resolution. In particular, we investigate the angular and time dependencies of these fluctuations, define appropriate correlation functions that reveal the fundamentally out of equilibrium nature of the observed fluctuations as well as their global anisotropy. Langevin equations respecting the symmetry of the system allow us to model the damped oscillatory behaviour of the time correlations.     

Manipulation of live mouse embryonic stem cells using holographic optical tweezers

We report the ability to move and arrange patterns of live embryonic stem cells using holographic optical tweezers. Single cell suspensions of mouse embryonic stem cells were manipulated with holographic optical tweezers into a variety of patterns including lines, curves and circles. Individual cells were also lifted out of the sample plane highlighting the potential for 3D positional control. Trypan blue dye exclusion and Live/Dead? staining (CMFDA^?1, EthHD^?1) showed that the cells were still viable after manipulation with the optical tweezers. The ability to move individual stem cells into specific, pre-defined patterns provides a method to study how arrangement and associated small-scale interactions occur between neighbouring cells.     

Influence of viscosity on chondrogenic differentiation of mesenchymal stem cells during 3D culture in viscous gelatin solution-embedded hydrogels

Differentiation of human bone marrow-derived mesenchymal stem cells(hMSCs)is regulated by a variety of cues of their surrounding microenvironments.In particular,mechanical properties of cell culture matrices have been recently disclosed to play a pivotal role in stem cell differentiation.However,it remains elusive how viscosity affects the chondrogenic differentiation of hMSCs during three-dimensional(3 D)culture.In this study,a 3 D culture system that was established by embedding viscous gelatin solution in chemically cross-linked gelatin hydrogels was used for 3 D culture of hMSCs in gelatin solutions with different viscosities.The influence of solution viscosity on chondrogenic differentiation of hMSCs was investigated.Viscous gelatin solutions promoted cell proliferation in the order of low,middle and high viscosity while elastic hydrogels restricted cell proliferation.High viscosity gelatin solution led to increased production of the cartilaginous matrix.Under the synergistic stimulation of chondrogenic induction factors,high viscosity was beneficial for the chondrogenic differentiation of hMSCs.The results suggested the role of viscosity should be considered as one of the dominant mechanical cues affecting stem cell differentiation.     

花鲈胚胎干细胞移植及嵌合体的构建

旨在通过花鲈胚胎干细胞(LJES1)移植构建嵌合体,证实LJES1细胞的体内分化能力和发育全能性.采用脂质体介导法将线性化pCMV-EGFP质粒导入到长期培养的LJES1细胞内,细胞绿色荧光蛋白(GFP)的表达率为5%～10%,以此为供体,通过显微注射方法把20～50个LJES1细胞移植到花鲈囊胚中,共移植囊胚478枚,获得了20枚表达GFP的嵌合体胚胎,其中的15枚胚胎发育成鱼苗.荧光显微观察和PCR检测显示了LJES1细胞可在宿主胚胎内片状嵌合和单细胞分散嵌合,嵌合部位可分布于胚体的三个胚层;同时也证明GFP是一种优良的遗传标记.本实验结果为证实LJES1细胞的发育全能性提供了有力证据.     

Neurogenic differentiation of human umbilical cord mesenchymal stem cells on aligned electrospun polypyrrole/polylactide composite nanofibers with electrical stimulation

Adult central nervous system (CNS) tissue has a limited capacity to recover after trauma or disease. Recent medical cell therapy using polymeric biomaterialloaded stem cells with the capability of differentiation to specific neural population has directed focuses toward the recovery of CNS. Fibers that can provide topographical, biochemical and electrical cues would be attractive for directing the differentiation of stem cells into electro-responsive cells such as neuronal cells. Here we report on the fabrication of an electrospun polypyrrole/polylactide composite nanofiber film that direct or determine the fate of mesenchymal stem cells (MSCs), via combination of aligned surface topography, and electrical stimulation (ES). The surface morphology, mechanical properties and electric properties of the film were characterized. Comparing with that on random surface film, expression of neurofilament-lowest and nestin of human umbilical cord mesenchymal stemcells (huMSCs) cultured on film with aligned surface topography and ES were obviously enhanced. These results suggest that aligned topography combining with ES facilitates the neurogenic differentiation of huMSCs and the aligned conductive film can act as a potential nerve scaffold.     

Collagen gel three-dimensional matrices combined with adhesive proteins stimulate neuronal differentiation of mesenchymal stem cells

Three-dimensional gel matrices provide specialized microenvironments that mimic native tissues and enable stem cells to grow and differentiate into specific cell types. Here, we show that collagen three-dimensional gel matrices prepared in combination with adhesive proteins, such as fibronectin (FN) and laminin (LN), provide significant cues to the differentiation into neuronal lineage of mesenchymal stem cells (MSCs) derived from rat bone marrow. When cultured within either a three-dimensional collagen gel alone or one containing either FN or LN, and free of nerve growth factor (NGF), the MSCs showed the development of numerous neurite outgrowths. These were, however, not readily observed in two-dimensional culture without the use of NGF. Immunofluorescence staining, western blot and fluorescence-activated cell sorting analyses demonstrated that a large population of cells was positive for NeuN and glial fibrillary acidic protein, which are specific to neuronal cells, when cultured in the three-dimensional collagen gel. The dependence of the neuronal differentiation of MSCs on the adhesive proteins containing three-dimensional gel matrices is considered to be closely related to focal adhesion kinase (FAK) activation through integrin receptor binding, as revealed by an experiment showing no neuronal outgrowth in the FAK-knockdown cells and stimulation of integrin β1 gene. The results provided herein suggest the potential role of three-dimensional collagen-based gel matrices combined with adhesive proteins in the neuronal differentiation of MSCs, even without the use of chemical differentiation factors. Furthermore, these findings suggest that three-dimensional gel matrices might be useful as nerve-regenerative scaffolds.     

Promotion of osteogenic differentiation of stem cells and increase of bone-bonding ability in vivo using urease-treated titanium coated with calcium phosphate and gelatin

Abstract

Because of its excellent biocompatibility and low allergenicity, titanium has been widely used for bone replacement and tissue engineering. To produce a desirable composite with enhanced bone response and mechanical strength, in this study bioactive calcium phosphate (CaP) and gelatin composites were coated onto titanium (Ti) via a novel urease technique. The cellular responses to the CaP/gelatin/Ti (CaP/gel/Ti) and bone bonding ability were evaluated with proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) on CaP/gel/Ti and CaP/Ti in vitro. The results showed that the optical density values, alkaline phosphatase expression and genes expression of MSCs on CaP/gel/Ti were similar to those on CaP/Ti, yet significantly higher than those on pure Ti (p < 0.05). CaP/gel/Ti and CaP/Ti rods (2 mm in diameter, 10 mm in length) were also implanted into femoral shaft of rabbits and pure Ti rods served as control (n = 10). Histological examination, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) measurements were performed at 4 and 8 weeks after the operation. The histological and SEM observations demonstrated clearly that more new bone formed on the surface of CaP/gel/Ti than in the other two groups at each time point. The CaP/gel/Ti bonded to the surrounding bone directly with no intervening soft tissue layer. An interfacial layer, containing Ti, Ca and P, was found to form at the interface between bone and the implant on all three groups by EDS analysis. However, the content of Ca, P in the surface of CaP/gel/Ti implants was more than in the other two groups at each time point. The CaP/gel/Ti modified by the urease method was not only beneficial for MSCs proliferation and osteogenic differentiation, but also favorable for bone bonding ability on Ti implants in vivo, suggesting that Ti functionalized with CaP and gelatin might have a great potential in clinical joint replacement or dental implants.     

3D culturing of human adipose-derived stem cells enhances their pluripotency and differentiation abilities

Human mesenchymal stem cells,such as human adipose-derived stem cells(hASCs),are typically cultured on a two-dimensional(2 D)monolayer material surface,on which 2 D culturing methods are easily performed and time-saving.However,hASCs usually suffer from decreased pluripotency and differentiation ability when cultured with a 2 D monolayer culturing method compared to hASCs cultured with a three-dimensional(3 D)culturing method,such as suspension culture.In this study,we evaluated whether the pluripotency and differentiation ability of hASCs can be reversibly changed during sequential cultivation with 2 D and 3 D culturing processes.The hASCs cultivated with a 3 D culturing process after 2 D culture showed at least 2-fold enhanced pluripotency(Sox2,Nanog,and OCT4)compared with that of hASCs cultured with the 2 D culture process alone.Furthermore,hASCs obtained from the 3 D culture process expressed increased levels of differentiation markers of chondrocytes and osteoblasts compared with hASCs obtained from the 2 D culture process when hASCs were induced to differentiate.However,their pluripotency and differentiation ability were extensively reduced when hASCs were shifted from 3 D culture to 2 D culture and vice versa,which indicates that hASCs show reversibility in terms of their pluripotency and differentiation ability depending on their environment in 2 D and 3 D culture.The reversibility of pluripotency and differentiation ability were found to last for at least 5 passages in culture during the alternative and sequential culture of cells with 2 D and 3 D culturing processes.Our study revealed the importance of the culture microenvironment in maintaining the pluripotency and differentiation ability of hASCs,which may reduce the effects of the aging process in hASCs.We discuss whether the environment of stem cell culture(i.e.,2 D or 3 D cultivation)can affect stem cell fate in terms of pluripotency and differentiation reversibility.     

The sensitivity of human mesenchymal stem cells to vibration and cold storage conditions representative of cold transportation

In the current study, the mechanical and hypothermic damage induced by vibration and cold storage on human mesenchymal stem cells (hMSCs) stored at 2–8°C was quantified by measuring the total cell number and cell viability after exposure to vibration at 50 Hz (peak acceleration 140 m s⁻² and peak displacement 1.4 mm), 25 Hz (peak acceleration 140 m s⁻², peak displacement 5.7 mm), 10 Hz (peak acceleration 20 m s⁻², peak displacement 5.1 mm) and cold storage for several durations. To quantify the viability of the cells, in addition to the trypan blue exclusion method, the combination of annexin V-FITC and propidium iodide was applied to understand the mode of cell death. Cell granularity and a panel of cell surface markers for stemness, including CD29, CD44, CD105 and CD166, were also evaluated for each condition. It was found that hMSCs were sensitive to vibration at 25 Hz, with moderate effects at 50 Hz and no effects at 10 Hz. Vibration at 25 Hz also increased CD29 and CD44 expression. The study further showed that cold storage alone caused a decrease in cell viability, especially after 48 h, and also increased CD29 and CD44 and attenuated CD105 expressions. Cell death would most likely be the consequence of membrane rupture, owing to necrosis induced by cold storage. The sensitivity of cells to different vibrations within the mechanical system is due to a combined effect of displacement and acceleration, and hMSCs with a longer cold storage duration were more susceptible to vibration damage, indicating a coupling between the effects of vibration and cold storage.