Synthesis of tethered-polymer brush by atom transfer radical polymerization from a plasma-polymerized-film-coated quartz crystal microbalance and its application for immunosensors

This study synthesizes a tethered surface-grafted poly(acrylic acid) with quartz crystal microbalance (QCM) surfaces and provides detailed analysis of their properties and application. A tethered polyelectrolyte brush of poly(acrylic acid) is generated by first covering the substrate with a plasma-polymerized allyl alcohol (pp-AA) film, changing the polymerization initiators (bromination), and then grafting through atom transfer radical polymerization (ATRP) of tert-butyl acrylate (t-BA); these initiators are immobilized on a surface and exposed to a monomer. Finally, we convert the poly(t-BA) brush into poly(acrylic acid) through hydrolysis. We use the QCM technique to measure configuration change of the tethered poly(acrylic acid) grafted chains with two different degrees of polymerization (DP=50,200) in aqueous solutions at three different pH values (4.0, 4.8, and 5.4). The tethered poly(acrylic acid) grafted QCM shows that repeatable frequency responses are induced by pH change of solution. These frequency responses of large DP for pH are 20 times larger than responses of lower DP for pH. The frequency response of antibody immobilization on tethered poly(acrylic acid) grafted QCM (DP=200) and its frequency response of immunoreaction are 10 times larger than conventional immobilization methods by cysteamine with glutalaldehyde coupling of the antibody. The tethered poly(acrylic acid) grafted QCM can increase the frequency response for pH, the immobilization amount of antibody, and immunosensor response.     

Rational design of cytophilic and cytophobic polyelectrolyte multilayer thin films

Nanostructured polyelectrolyte multilayer thin films electrostatically assembled alternately from such polymers as poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were investigated for their in vitro cell interactions. Not surprisingly, NR6WT cells, a highly adhesive murine fibroblast cell line, attached to many different multilayer combinations tested. However, PAH/PAA multilayers constructed at pH deposition conditions of 2.0/2.0 were completely bioinert. Analogous cell interactions were observed with PAH/poly(methacrylic acid) (PAH/PMA), PAH/sulfonated poly(styrene) (PAH/SPS), and poly(diallyldimethylammonium chloride)/SPS (PDAC/SPS) systems, thereby suggesting a general trend in the fibroblasts' response to multilayers. Specifically, highly ionically stitched films attracted cells, whereas weakly ionically cross-linked multilayers, which swell substantially in physiological conditions to present richly hydrated surfaces, resisted fibroblast attachment. Thus, by manipulating the multilayer pH or ionic strength assembly conditions or both, which in turn dictate the molecular architecture of the thin films, one may powerfully direct a single multilayer combination to be either cell adhesive or cell resistant.     

Neurogenesis of bone marrow-derived mesenchymal stem cells onto β-mercaptoethanol-loaded PLGA film

Bone marrow-derived mesenchymal stem cells (BMSCs) are of particular interest in the field of tissue engineering because of their potential to differentiate into osteoblasts, chondrocytes, and neuronal cells. In order to promote the differentiation of BMSCs into specific cell types, appropriate scaffold biomaterials and bioactive molecules that can support the differentiation of BMSCs into specific cell types are needed. We hypothesized that β-mercaptoethanol (BME), which has been reported to induce the differentiation of BMSCs into neural-like cells, promotes BMSCs to differentiate into neural-like cells when BME is added to polymeric scaffolds containing the BMSCs. We fabricated biocompatible film shaped scaffolds composed of poly(lacti-co-glycolic) acid (PLGA) and various concentrations of BME to confirm that BME-promoted differentiation of BMSCs is concentration-dependent. Cell proliferation increased as the BME concentration in the films increased at the early stage, and the proliferation rate remained similar on the PLGA films for 3 weeks following the BMSC seeding. The expression of neuronal markers in differentiated BMSCs was assessed by RT-PCR. At 2- and 3-week time-points, mRNA expression of neurofilament and neuron specific enolase was significantly increased in PLGA/BME films containing 400 μM BME compared to PLGA films. Thus, we have identified BMSC-seeded PLGA/BME films with 200 μM and 400 μM BME as potentially useful candidates for neural tissue engineering applications by promoting BMSC proliferation and differentiation towards neural-like cells.     

动物胚胎干细胞诱导分化的研究进展

胚胎干细胞 (ES细胞 )是从动物早期胚胎的内细胞团或原始生殖细胞分离出来的具有发育全能性的一种未分化的无限增殖细胞系 ,ES细胞能体外诱导分化为神经细胞、肌肉细胞、成纤维细胞等各种细胞。综述了动物的ES细胞的分化诱导机理及目前体外诱导分化的研究现状     

Recovery from Parkinsonism with N-acetylcysteine-differentiated neurons

The upregulation of dopaminergic neuronal differentiation is necessary for stem cell therapy in Parkinson’s disease (PD). In this study, neuronal differentiation efficiency increased by more than 2 times in P19 embryonic stem cells (ESCs) induced by N-acetylcysteine (NAC) and retinoic acid (RA) as compared to RA alone, with suppressed glial differentiation. The majority of NAC-treated stem cells grafted into brains of PD mice differentiated into dopaminergic neurons and persisted well for 6 weeks. Parkinsonism was also greatly improved after grafting NAC-treated cells in comparison to cells treated with only RA. Our results strongly suggest that NAC treatment may be an effective strategy for generating stem cells fated to become dopaminergic neurons for PD clinical therapy.     

Modulation of spreading, proliferation, and differentiation of human mesenchymal stem cells on gelatin-immobilized poly(L-lactide-co--caprolactone) substrates

Controlled adhesion and continuous growth of human mesenchymal stem cells (hMSCs) are essential for scaffold-based delivery of hMSCs in tissue engineering applications. The main goal of this study is to develop biofunctionalized synthetic substrates to actively control adhesion, spreading, and proliferation of hMSCs. gamma-Ray irradiation was employed to graft acrylic acid (AAc) to biodegeradable poly(L-lactide-co--caprolactone) (PLCL) films. Gelatin, a natural polymer, was then immobilized on the AAc grafted PLCL film (AAc-PLCL) to induce biomimetic interactions with the cells. The graft yield of AAc increased as the irradiation dose and AAc concentration increased, and the presence of gelatin (gelatin-AAc-PLCL) following immobilization was confirmed using ESCA. To investigate cell responses, hMSCs isolated from a human mandible were cultured on the various substrates and their adhesion, spreading, and proliferation were examined. After three days of culture, the DNA concentration from the cells cultured on gelatin-AAc-PLCL film was 2.9-fold greater than that on the PLCL film. Immunofluorescent staining of hMSCs cultured on the gelatin-AAc-PLCL films demonstrated homogeneous localization of F-Actin and vinculin in their cytoplasm, while mature adhesive structure was not observed from the cells cultured on other substrates. Furthermore, the ratio of projected area of adherent single cells on gelatin-AAc-PLCL films was significantly larger (116.80 +/- 12.78%) than that on the PLCL films (30.11 +/- 5.07%). Our results suggest that gelatin-immobilized PLCL substrates may be potentially used in tissue engineering, particularly as a stem cell delivery carrier for the regeneration of target tissue.     

Micropatterned matrix directs differentiation of human mesenchymal stem cells towards myocardial lineage

Stem cell response can be influenced by a multitude of chemical, topological and mechanical physiochemical cues. While extensive studies have been focused on the use of soluble factors to direct stem cell differentiation, there are growing evidences illustrating the potential to modulate stem cell differentiation via precise engineering of cell shape. Fibronectin were printed on poly(lactic-co-glycolic acid) (PLGA) thin film forming spatially defined geometries as a means to control the morphology of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs that were cultured on unpatterned substrata adhered and flattened extensively (∼ 10,000 μm²) while cells grown on 20 μm micropatterend wide adhesive strips were highly elongated with much smaller area coverage of ∼ 2000 μm². Gene expression analysis revealed up-regulation of several hallmark markers associated to neurogenesis and myogenesis for cells that were highly elongated while osteogenic markers were specifically down-regulated or remained at its nominal level. Even though there is clearly upregulated levels of both neuronal and myogenic lineages but at the functionally relevant level of protein expression, the myogenic lineage is dominant within the time scale studied as determined by the exclusive expression of cardiac myosin heavy chain for the micropatterned cells. Enforced cell shape distortion resulting in large scale rearrangement of cytoskeletal network and altered nucleus shape has been proposed as a physical impetus by which mechanical deformation is translated into biochemical response. These results demonstrated for the first time that cellular shape modulation in the absence of any induction factors may be a viable strategy to coax lineage-specific differentiation of stem cells.     

神经干细胞脑内移植后的存活、迁移和分化

从胚胎或成体大鼠脑组织、人胚脑组织均能分离到神经干细胞 ,将它们进行体外原代培养扩增或永生化后植入脑内 ,均能观察到其在脑内的迁移和分化现象。其分化能力主要取决于移植部位的脑内微环境 ,但这种影响作用是相对的。同时 ,体外培养环境如培养时间和细胞融合程度、维甲酸类诱导分化剂处理、NGF转导处理再移植或与嗜铬细胞 (分泌NGF)共移植等 ,也能决定神经干细胞脑内移植后向神经元方向分化的能力。神经干细胞移植为中枢神经系统功能重建和神经再生带来新的希望。     

Efficient differentiation of mouse embryonic stem cells into motor neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9. Using this robust protocol, we achieved 51 ± 0.8% of differentiation efficiency (n = 3; p < 0.01, Student's t-test). Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.     

Micropatterning of polymer brushes: grafting from dewetting polymer films for biological applications

In this novel platform, a micropatterned polymer brush was obtained by grafting poly(poly(ethylene glycol) methyl ether methacrylate) (poly(PEGMA)) from a thin macroinitiator film using atom transfer radical polymerization (ATRP). A pattern of holes was formed in the macroinitiator film by taking advantage of its spontaneous dewetting above the glass transition temperature from a bottom polystyrene film, driven by unfavorable intermolecular forces. Patterning by dewetting can be achieved at length-scales from a few hundred nanometers to several tens of micrometers, by simply thermally annealing the bilayer above the glass transition temperature of the polymer. This approach is substrate-independent, as polymer films can be cast onto surfaces of different size, shape, or material. As a demonstration of its potential, proteins, and individual cells were attached on targeted bioadhesive polystyrene areas of the micropatterns within poly(PEGMA) protein-repellent brushes. We anticipate this approach will be suitable for the patterning of brushes, especially for biomedical applications such as in the study of single cells and of cell cocultures.     

Fibronectin and cell attachment to cell and protein resistant polyelectrolyte surfaces

Culture of A7r5 smooth muscle cells on a polyelectrolyte multilayer film (PEMU) can influence various cell properties, including adhesion, motility, and cytoskeletal organization, that are modulated by the extracellular matrix (ECM) in vivo. ECM contribution to cell behavior on PEMUs was investigated by determining the amount of fibronectin (FN) bound to charged and hydrophobic PEMUs by optical waveguide lightmode spectroscopy and immunofluorescence microscopy. FN bound best to poly(allylamine hydrochloride) (PAH)-terminated and Nafion-terminated PEMUs. FN bound poorly to PEMUs terminated with a copolymer of poly(acrylic acid) (PAA) and 3-[2-(acrylamido)-ethyl dimethylammonio] propane sulfonate (PAA-co-AEDAPS). Cells adhered and spread well on the Nafion-terminated PEMU surfaces. In contrast, cells spread less and migrated more on both FN-coated and uncoated PAH-terminated PEMU surfaces. Both cells and FN interacted much better with Nafion than with PAA-co-PAEDAPS in a micropatterned PEMU. These results indicate that A7r5 cell adhesion, spreading, and motility on PEMUs can be independent of FN binding to the surfaces.     

Embryonic Stem Cells: Spontaneous and Directed Differentiation

The specific structural features of embryonic stem cells and embryoid bodies and mechanisms of their differentiation in different cell types are considered. The mouse embryonic stem cells (line R1) formed multilayer colonies which enlarged as a result of fast cell division. Embryoid bodies that derived from embryonic stem cells consisted of an outer layer, an inner layer, and an internal cavity. The structure of cells of the outer and inner layers markedly differed. Spontaneous and directed differentiation of embryoid bodies is determined by some unspecific and specific factors (growth and differentiation factors and extracellular matrix proteins). Retinoic acid, the most commonly used inducer of differentiation of the embryonic stem cells, induces different types of differentiation when applied at different concentrations. The sequence of expression of tissue specific genes and proteins during differentiation of the embryonic stem cells in vitrois similar to that in vivo.     

Interspecies chimeric conditions affect the developmental rate of human pluripotent stem cells

Human pluripotent stem cells hold significant promise for regenerative medicine. However, long differentiation protocols and immature characteristics of stem cell-derived cell types remain challenges to the development of many therapeutic applications. In contrast to the slow differentiation of human stem cells in vitro that mirrors a nine-month gestation period, mouse stem cells develop according to a much faster three-week gestation timeline. Here, we tested if co-differentiation with mouse pluripotent stem cells could accelerate the differentiation speed of human embryonic stem cells. Following a six-week RNA-sequencing time course of neural differentiation, we identified 929 human genes that were upregulated earlier and 535 genes that exhibited earlier peaked expression profiles in chimeric cell cultures than in human cell cultures alone. Genes with accelerated upregulation were significantly enriched in Gene Ontology terms associated with neurogenesis, neuron differentiation and maturation, and synapse signaling. Moreover, chimeric mixed samples correlated with in utero human embryonic samples earlier than human cells alone, and acceleration was dose-dependent on human-mouse co-culture ratios. The altered gene expression patterns and developmental rates described in this report have implications for accelerating human stem cell differentiation and the use of interspecies chimeric embryos in developing human organs for transplantation.     

miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells

The use of pluripotent stem cells in regenerative medicine and disease modeling is complicated by the variation in differentiation properties between lines. In this study, we characterized 13 human embryonic stem cell (hESC) and 26 human induced pluripotent stem cell (hiPSC) lines to identify markers that predict neural differentiation behavior. At a general level, markers previously known to distinguish mouse ESCs from epiblast stem cells (EPI-SCs) correlated with neural differentiation behavior. More specifically, quantitative analysis of miR-371-3 expression prospectively identified hESC and hiPSC lines with differential neurogenic differentiation propensity and in vivo dopamine neuron engraftment potential. Transient KLF4 transduction increased miR-371-3 expression and altered neurogenic behavior and pluripotency marker expression. Conversely, suppression of miR-371-3 expression in KLF4-transduced cells rescued neural differentiation propensity. miR-371-3 expression level therefore appears to have both a predictive and a functional role in determining human pluripotent stem cell neurogenic differentiation behavior.     

Porous poly (L-lactic acid) scaffolds are optimal substrates for internal colonization by A6 mesoangioblasts and immunocytochemical analyses

In this study, mouse mesoangioblasts were seeded onto bidimensional matrices within three-dimensional porous scaffolds of poly (L-lactic acid) (PLLA), in the presence or absence of a type I collagen coating. The cells were observed under a scanning electron microscope and tested for their adhesion, survival and proliferation. Immunolocalization of heat shock protein (Hsp) 70, an abundant and ubiquitous intracellular protein in these cells, was also performed in sectioned cell-containing scaffolds under a confocal fluorescence microscope to determine if in situ analysis of intracellular constituents was feasible. The data show that PLLA films allow direct cell adhesion and represent an optimal support for cell growth, and that the internal surfaces of PLLA polymeric sponges can be colonized by mesoangioblasts, which can be submitted for in situ confocal microscopic analyses for possible monitoring of time-dependent expression of differentiation markers.     

Addition of biological functionality to poly(epsilon-caprolactone) films

Biodegradable polyesters such as poly(epsilon-caprolactone) (PCL) have a number of biomedical applications; however, their usage is often limited by a lack of biological functionality. In this paper, a PCL-based polymer containing pendent groups activated by 4-nitrophenyl chloroformate (NPC) and reactive toward primary amines has been cast into thin films. The reactivity of the films toward poly(l-lysine) and the cell adhesion peptide, GRGDS, was assessed, and their cell adhesive capabilities were characterized. ATR-FTIR analysis found that NPC functional groups were present on the surface of the cast film, and the synthesis, conjugation, and visualization of a fluorescent molecule on these films further demonstrated the success of this functionalization methodology. The immersion of these films into a solution of either poly(l-lysine) (PLL) or GRGDS in PBS (pH 7.4) and subsequent 3T3 fibroblast adhesion studies demonstrated significant improvement in cell adhesion and spreading over films cast from unmodified PCL. This investigation has shown that this novel NPC-containing polymer can be utilized in many applications where increased cellular adhesion is required, or the coupling of specific molecules to polymer surfaces is of interest.     

小鼠胚胎干细胞结合丝素材料向神经细胞分化的实验研究

以小鼠胚胎干细胞（ES）为种子细胞,使用改良的4-/4+ RA方案,诱导小鼠ES细胞在丝素材料上向神经细胞分化,探讨丝素材料对其生长、黏附、分化等情况的影响。将小鼠ES细胞悬浮培养4 d得到的拟胚体（EBs）分别接种到经丝素膜和明胶包被的培养皿上进行诱导,比较不同材料上EBs的贴壁率及向神经元分化的比率。结果表明EBs在明胶和柞蚕丝素蛋白膜（TSF）上贴壁较快,平均贴壁率为90.3%和84.4%,在桑蚕丝素蛋白膜（SF）上贴壁较慢,贴壁率低,仅为38.5%,同时三者神经元的分化比率均能达到40%以上,无明显差异。通过以上实验,我们得出,TSF有望成为小鼠ES细胞向神经细胞分化的支架材料。