Nanotechnology in the regulation of stem cell behavior

Abstract

Stem cells are known for their potential to repair damaged tissues. The adhesion, growth and differentiation of stem cells are likely controlled by the surrounding microenvironment which contains both chemical and physical cues. Physical cues in the microenvironment, for example, nanotopography, were shown to play important roles in stem cell fate decisions. Thus, controlling stem cell behavior by nanoscale topography has become an important issue in stem cell biology. Nanotechnology has emerged as a new exciting field and research from this field has greatly advanced. Nanotechnology allows the manipulation of sophisticated surfaces/scaffolds which can mimic the cellular environment for regulating cellular behaviors. Thus, we summarize recent studies on nanotechnology with applications to stem cell biology, including the regulation of stem cell adhesion, growth, differentiation, tracking and imaging. Understanding the interactions of nanomaterials with stem cells may provide the knowledge to apply to cell–scaffold combinations in tissue engineering and regenerative medicine.     

Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

Abstract

While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell–cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examine the role of matrix rigidity on cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using a genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.     

具有区间摄动鲁棒性的调度问题

本文讨论了目标函数为折扣加权总完工时间的调度问题。对调度问题中最优调度关于加工时间在某一闭区间上任意变化时的不变性进行了讨论,得出了当加工时间在某一闭区间上变化时最优调度不变的充要条件。     

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.     

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.     

Analysis of symmetric domains in advanced applications with the boundary element method

The strategy in the boundary element method for the analysis of symmetric domains that does not require the modelling of contours corresponding to the axes of symmetry is investigated for a number of advanced applications. These applications include: treatment of domain loadings, two-dimensional time domain transient elastodynamics, and the analysis of probabilistic problems in elastostatics with a random geometric configuration. Both symmetric as well as arbitrary loadings acting on the symmetric objects are considered. A number of case studies are presented to provide comparisons of computer memory and CPU time requirements for the analyses of the entire object versus the analyses of only the symmetric portion of the object. The numerical data presented clearly demonstrates the advantages of developing efficient symmetric formulations for advanced applications.     

Osteoblast and stem cell response to nanoscale topographies: a review

To understand how cells respond to the nanoscale extracellular environment in vivo, cells from various sources have been cultured on nanoscale patterns fabricated using bottom-up and top-down techniques. Human fetal osteoblasts (hFOBs) and stem cells are some of them and they are known to be overtly responsive to nanoscale topographies – allowing us to investigate the hows and whys of the response in vitro. Information gathered from these in vitro studies could be used to control the cells, i.e. make the stem cells differentiate or retain their characteristics without the use of medium supplements. In this review, hFOB and stem cell responses to nanotopographies are summarized and discussed to shed some light on the influence of patterns on the reactions. Although both types of cells are responsive to nanoscale topographies, the responses are found to be unique to topographical dimension, shape, orientation and the types of cells used. This implies that cellular responses are influenced by multitude of factors and that if done right, cheaper self-assembled nanotopographies can be tailored to control the cells. A new self-assembly, powder-based technique is also included to provide an insight into the future of nanofabrication.     

Spatial stochastic modelling of the Hes1 gene regulatory network: intrinsic noise can explain heterogeneity in embryonic stem cell differentiation

Individual mouse embryonic stem cells have been found to exhibit highly variable differentiation responses under the same environmental conditions. The noisy cyclic expression of Hes1 and its downstream genes are known to be responsible for this, but the mechanism underlying this variability in expression is not well understood. In this paper, we show that the observed experimental data and diverse differentiation responses can be explained by a spatial stochastic model of the Hes1 gene regulatory network. We also propose experiments to control the precise differentiation response using drug treatment.     

Fluctuations in T cell receptor and pMHC interactions regulate T cell activation

Adaptive immune responses depend on interactions between T cell receptors (TCRs) and peptide major histocompatibility complex (pMHC) ligands located on the surface of T cells and antigen presenting cells (APCs), respectively. As TCRs and pMHCs are often only present at low copy numbers their interactions are inherently stochastic, yet the role of stochastic fluctuations on T cell function is unclear. Here, we introduce a minimal stochastic model of T cell activation that accounts for serial TCR-pMHC engagement, reversible TCR conformational change and TCR aggregation. Analysis of this model indicates that it is not the strength of binding between the T cell and the APC cell per se that elicits an immune response, but rather the information imparted to the T cell from the encounter, as assessed by the entropy rate of the TCR-pMHC binding dynamics. This view provides an information-theoretic interpretation of T cell activation that explains a range of experimental observations. Based on this analysis, we propose that effective T cell therapeutics may be enhanced by optimizing the inherent stochasticity of TCR-pMHC binding dynamics.     

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.     

视线跟踪系统角膜曲率中心模型优化算法及鲁棒性

为了高效获取3D视线跟踪技术中的角膜曲率中心,使系统满足实时准确及稳定性需求,本文构建了两类角膜曲率中心模型及其求解方法．首先,利用光学原理及眼球特征建立了两种求解该中心的模型,即非线性方程组模型和改进的模型．其次,针对模型特点构造了一种基于遗传算法与LM算法的新型混合算法（GA-LM）,以快速求解所建模型．最后,多组数值实验结果表明本文所建立的模型及GA-LM算法是有效的,其能快速准确求出3D视线跟踪系统中的角膜曲率中心．     

Poly(lactic acid) porous scaffold with calcium phosphate mineralized surface and bone marrow mesenchymal stem cell growth and differentiation

This work aims to modify the surface of a poly(lactic acid) (PLA) porous scaffold with calcium phosphate (CaP) via a simple solution-based technique, and to evaluate the effects of this modification on the responses of rat bone marrow mesenchymal stem cells (rBMMSCs). Under appropriate modification conditions involving stepwise-treatments in the Ca-and-P supersaturated solution under gentle agitation, a thin, poorly crystallized CaP layer was deposited. The BMMSCs derived from adult rats were shown to adhere quite well to the CaP-coated scaffold, and to proliferate actively with culturing time, although some down-regulation was noted with regard to the unmodified PLA scaffold. The osteogenic differentiation of rBMMSCs was significantly higher on the CaP-modified scaffold than on the unmodified scaffold, as confirmed by alkaline phosphatase (ALP) activity. Moreover, the expression of genes associated with bone, including collagen type I, osteopontin and bone sialoprotein, was stimulated better on the CaP-modified PLA scaffold. Based on these results, the currently used CaP-treatment was deemed effective in stimulating the osteogenic development of rBMMSCs on the PLA-based scaffold, and the CaP-treated PLA scaffold may be useful for future bone tissue engineering.     

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.     

The effects of vibration loading on adipose stem cell number,viability and differentiation towards bone-forming cells

Mechanical stimulation is an essential factor affecting the metabolism of bone cells and their precursors. We hypothesized that vibration loading would stimulate differentiation of human adipose stem cells (hASCs) towards bone-forming cells and simultaneously inhibit differentiation towards fat tissue. We developed a vibration-loading device that produces 3g peak acceleration at frequencies of 50 and 100 Hz to cells cultured on well plates. hASCs were cultured using either basal medium (BM), osteogenic medium (OM) or adipogenic medium (AM), and subjected to vibration loading for 3 h d^–1 for 1, 7 and 14 day. Osteogenesis, i.e. differentiation of hASCs towards bone-forming cells, was analysed using markers such as alkaline phosphatase (ALP) activity, collagen production and mineralization. Both 50 and 100 Hz vibration frequencies induced significantly increased ALP activity and collagen production of hASCs compared with the static control at 14 day in OM. A similar trend was detected for mineralization, but the increase was not statistically significant. Furthermore, vibration loading inhibited adipocyte differentiation of hASCs. Vibration did not affect cell number or viability. These findings suggest that osteogenic culture conditions amplify the stimulatory effect of vibration loading on differentiation of hASCs towards bone-forming cells.