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.     

Embryonic Stem Cells Biosensor and Its Application in Drug Analysis and Toxin Detection

To investigate the use of stem cells as biosensor elements, a novel cell-based light-addressable potentiometric sensor (LAPS) was developed for monitoring cellular beating. Mouse embryonic stem cells were induced to differentiate into cardiomyocytes in vitro. Extracellular field potentials of spontaneously beating cardiomyocytes induced from stem cells were recorded by LAPS in the potential and frequency ranges of 25-45 muV and 0.5-3 Hz, respectively. Due to its capability of monitoring important physiological parameters such as potential and frequency in vitro, the sensor can be used in drug analysis and toxin detection in a long-term and noninvasive way. The pharmacological and toxicological researches make it possible to use stem cells-based biosensor for biomedical assays.     

In situ spectral monitoring of mRNA translation in embryonic stem cells during differentiation in vitro

Raman microspectroscopy was used to determine biochemical markers during the differentiation of embryonic murine stem cells (mES) in vitro. Such markers are useful to determine the differentiation status of ES cells cultured on biomaterials. Raman spectra of mES cells as undifferentiated, spontaneously differentiated (4 days), and differentiated cells via formation of embryoid bodies (16, 20 days) were analyzed. Unsupervised hierarchical cluster analysis and principal component analysis were used to determine biochemical differences between mES cells in various states of differentiation. The undifferentiated cells were characterized by high scores of the first principal component (PC1, 49% variance). Similarity between the PC1 loading and the Raman spectrum of RNA indicated a high concentration of RNA in mES cells compared to differentiated cells. The ratio between the peak areas of RNA and proteins was used as a measure of mRNA translation. Using the same peak area ratio, it was possible to differentiate even between mES as undifferentiated and in early stages of differentiation (4 days). These findings were correlated with biological studies reporting high levels of nontranslated mRNA during early embryonic development. Therefore, the RNA translation obtained from the Raman spectra can be used as marker of differentiation state of mES cells.     

Electroconductive Biohybrid Collagen/Pristine Graphene Composite Biomaterials with Enhanced Biological Activity

Electroconductive substrates are emerging as promising functional materials for biomedical applications. Here, the development of biohybrids of collagen and pristine graphene that effectively harness both the biofunctionality of the protein component and the increased stiffness and enhanced electrical conductivity (matching native cardiac tissue) obtainable with pristine graphene is reported. As well as improving substrate physical properties, the addition of pristine graphene also enhances human cardiac fibroblast growth while simultaneously inhibiting bacterial attachment (Staphylococcus aureus). When embryonic‐stem‐cell‐derived cardiomyocytes (ESC‐CMs) are cultured on the substrates, biohybrids containing 32 wt% graphene significantly increase metabolic activity and cross‐striated sarcomeric structures, indicative of the improved substrate suitability. By then applying electrical stimulation to these conductive biohybrid substrates, an enhancement of the alignment and maturation of the ESC‐CMs is achieved. While this in vitro work has clearly shown the potential of these materials to be translated for cardiac applications, it is proposed that these graphene‐based biohybrid platforms have potential for a myriad of other applications—particularly in electrically sensitive tissues, such as neural and neural and musculoskeletal tissues.     

Nanocomposite Elastomeric Biomaterials for Myocardial Tissue Engineering Using Embryonic Stem Cell‐derived Cardiomyocytes

Regeneration or repair of the damaged myocardium requires different strategies including engineered constructs for more efficient cell delivery. This study was undertaken to examine the potential of a new nanostructured elastomer to deliver embryonic stem cell‐derived cardiomyocytes (ESC‐CM) to an infarcted area of the myocardium. Engineered materials were biocompatible, mechanically stable, and elastomeric nanocomposites serving as substrates for delivery of ESC‐CM and as a left ventricular support device in myocardial regeneration strategies. Materials investigated were soft and strong poly(aliphatic/aromatic‐ester) multiblock thermoplastic elastomers with poly(ethylene terephthalate) (PET) hard segments and dimerized fatty acid, i.e., dilinoleic acid (DLA) soft segments, respectively, with and without addition of 0.2 wt% TiO₂ nanoparticles to form nanocomposites. The PET/DLA‐TiO₂ nanocomposite exhibited over 8 MPa tensile strength and 900% elongation at break. Addition of TiO₂ nanoparticles significantly altered surface roughness and enhanced adhesion and spreading of ESC‐CM derived from mouse and human embryonic stem cells. The newly developed materials did not affect the functional activity of spontaneously beating hESC‐CM, as demonstrated by unaltered rate of their beating, and the cells continued to demonstrate contractile activity on the materials for more than two months in culture (the longest time tested). Quantitative proliferation and survival assays using fibroblasts confirmed the ability of the new materials to support cells as well as or better than the present commercial‐type thermoplastic elastomer analog. The results indicate that PET/DLA and PET/DLA‐TiO₂ are promising candidates for the manufacture of engineered patches to deliver ESC‐CMs.     

Substrate stiffness affects skeletal myoblast differentiation in vitro

Abstract

To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ε-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.     

Historic and current strategies in bone tissue engineering: Do we have a hope in Hench?

Professors Larry Hench and Julia Polak formed the Tissue Engineering and Regenerative Medicine Centre (TERM) at Imperial College London to foster collaborations between biologists and materials scientists. Early work at the center elucidated the biomolecular interactions between primary human osteoblasts and 45S5 Bioglass . As research efforts expanded, the team discovered that the dissolution products of both 45S5 Bioglass and 58S sol-gel bioactive glasses had osteoblastic stimulatory properties. To address the shortage of appropriate cells for bone tissue engineering applications, TERM scientists also demonstrated the differentiation of embryonic stem (ES) cells to osteoblasts when treated with the dissolution products of bioactive glasses. They also found that the soluble factors ascorbic acid, beta -glycerophosphate, and dexamethasone preferentially differentiated ES cells to osteoblasts, and their combination with the dissolution products of bioactive glasses stimulated differentiation even further. Taken together, these results demonstrate the suitability of bioactive glasses as scaffolds for bone tissue engineering as they not only provide an osteoconductive and osteoproductive substrate, but also actively stimulate cells to express appropriate osteoblastic phenotypes. Professor Hench's vision to pioneer regenerative medicine research continues with the aim of developing novel therapeutics to treat musculoskeletal disability.     

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.     

Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

Abstract

The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell–matrix interaction, using poly-ε-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.     

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.     

Remote Magnetic Nanoparticle Manipulation Enables the Dynamic Patterning of Cardiac Tissues

The ability to manipulate cellular organization within soft materials has important potential in biomedicine and regenerative medicine; however, it often requires complex fabrication procedures. Here, a simple, cost-effective, and one-step approach that enables the control of cell orientation within 3D collagen hydrogels is developed to dynamically create various tailored microstructures of cardiac tissues. This is achieved by incorporating iron oxide nanoparticles into human cardiomyocytes and applying a short-term external magnetic field to orient the cells along the applied field to impart different shapes without any mechanical support. The patterned constructs are viable and functional, can be detected by T₂*-weighted magnetic resonance imaging, and induce no alteration to normal cardiac function after grafting onto rat hearts. This strategy paves the way to creating customized, macroscale, 3D tissue constructs with various cell-types for therapeutic and bioengineering applications, as well as providing powerful models for investigating tissue behavior.     

Cardiomyocytes‐Actuated Morpho Butterfly Wings

Morpho butterflies are famous for their wings' brilliant structural colors arising from periodic nanostructures, which show great potential value for fundamental research and practical applications. Here, a novel cellular mechanical visualizable biosensor formed by assembling engineered cardiac tissues on the Morpho butterfly wings is presented. The assembled cardiomyocytes benefit from the periodic parallel nanoridges of the wings and can recover their autonomic beating ability with guided cellular orientation and good contraction performance. As the beating processes are accompanied by the cardiomyocytes' elongation and contraction, the elastic butterfly wing substrate undergoes the same cycle of deformations, which causes corresponding synchronous shifts in their structural colors and photonic bandgaps for self‐reporting of the cell mechanics. It is demonstrated that this self‐reporting performance can be further improved by adding oriented carbon nanotubes in the nanoridges of the wings for the culture. In addition, taking advantage of the similar size of the cardiomyocyte and a single Morpho wing scale, the investigation of single‐cell‐level mechanics can be realized by detecting the optical performance of a single scale. These remarkable properties make these butterfly wings ideal platforms for biomedical research.     

Substrate stiffness affects skeletal myoblast differentiation in vitro

To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ε-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.     

Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell–matrix interaction, using poly-ε-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.     

禽类胚胎干细胞的研究

综述了禽类胚胎干细胞的分离培养条件、生物学特性鉴定及应用展望。     

Soft fibrin gels promote selection and growth of tumorigenic cells

The identification of stem-cell-like cancer cells through conventional methods that depend on stem cell markers is often unreliable. We developed a mechanical method for selecting tumorigenic cells by culturing single cancer cells in fibrin matrices of ~100?Pa in stiffness. When cultured within these gels, primary human cancer cells or single cancer cells from mouse or human cancer cell lines grew within a few days into individual round colonies that resembled embryonic stem cell colonies. Subcutaneous or intravenous injection of 10 or 100 fibrin-cultured cells in syngeneic or severe combined immunodeficiency mice led to the formation of solid tumours at the site of injection or at the distant lung organ much more efficiently than control cancer cells selected using conventional surface marker methods or cultured on conventional rigid dishes or on soft gels. Remarkably, as few as ten such cells were able to survive and form tumours in the lungs of wild-type non-syngeneic mice.     

Application of Nanomaterials in Stem Cell-Based Therapeutics for Cardiac Repair and Regeneration

Cardiovascular disease is a leading cause of disability and death worldwide. Although the survival rate of patients with heart diseases can be improved with contemporary pharmacological treatments and surgical procedures, none of these therapies provide a significant improvement in cardiac repair and regeneration. Stem cell-based therapies are a promising approach for functional recovery of damaged myocardium. However, the available stem cells are difficult to differentiate into cardiomyocytes, which result in the extremely low transplantation efficiency. Nanomaterials are widely used to regulate the myocardial differentiation of stem cells, and play a very important role in cardiac tissue engineering. This study discusses the current status and limitations of stem cells and cell-derived exosomes/micro RNAs based cardiac therapy, describes the cardiac repair mechanism of nanomaterials, summarizes the recent advances in nanomaterials used in cardiac repair and regeneration, and evaluates the advantages and disadvantages of the relevant nanomaterials. Besides discussing the potential clinical applications of nanomaterials in cardiac therapy, the perspectives and challenges of nanomaterials used in stem cell-based cardiac repair and regeneration are also considered. Finally, new research directions in this field are proposed, and future research trends are highlighted.     

Biomaterials control of pluripotent stem cell fate for regenerative therapy

Pluripotent stem cells (PSCs) derived from either the embryo or reprogramming processes have the capacity to self-renew and differentiate into various cells in the body, thereby offering a valuable cell source for regenerative therapy of intractable disease and serious tissue damage. Traditionally, methods to expand and differentiate PSCs have been confined to 2D culture through the use of biochemical signals; the use of biomaterials beyond the commercially available culture dish has not been widespread. Nevertheless, biomaterials with tailored physical, chemical, and geometrical cues can mimic the native stem cell niche to tune the microenvironmental conditions for PSCs to preserve their self-renewal capacity or to switch their phenotype, a status ultimately needed to gain regenerative functions ex vivo and in vivo. Recently efforts to explore biomaterials to regulate PSC behavior have accelerated. The biomaterials properties investigated include surface chemistry, immobilized ligand, nano-/micro-topography, matrix stiffness, geometrical complexity, 3D configuration, and combinations thereof. This review aims to cover the current advances of biomaterials-based control over PSCs, particularly for the preservation of self-renewal capacity as well as for their differentiation into target cells. Furthermore, it aims to suggest future research directions that would facilitate the eventual translation of these advances.     

A chitosan-graft-PEI-eprosartan conjugate for cardiomyocyte-targeted VEGF plasmid delivery in myocardial ischemia gene therapy

A multifunctional copolymer conjugate chitosan-graft-polyethyleneimine-eprosartan (CPE) containing low-molecular-weight chitosan backbone and polyethyleneimine (PEI) arms with eprosartan (ES) conjugated via an amide bond was fabricated as a cardiomyocyte-targeted vascular endothelial growth factor (VEGF) plasmid delivery nanovector. Here, ES was utilised to specifically bind to overexpressed angiotensin II type 1 receptor (AT₁R) of cardiomyocytes and strengthen endosomal buffering capacity of CPE. The self-assembled CPE/VEGF complexes exhibited desirable and homogenous particle size, moderate positive charges, advanced gene condensation and protection profiles in vitro. Flow cytometry and ELISA analyses confirmed that ES-targeted function and ES-enhanced buffering capacity induced specific cellular uptake and high transfection of CPE/VEGF complexes in AT₁R-overexpressed H9C2 cells. In vivo investigation on myocardial ischemia rat model revealed that CPE/VEGF complexes possessed strong therapeutic effect. These findings suggested that CPE could be an ideal cardiomyocyte-targeted nanovector for effective transfer of VEGF plasmid, and a multifunctional CPE/VEGF delivery system with cardiomyocyte-specific targetability, enhanced endosomal buffering capacity and strong therapeutic effect might be a new promising strategy for effective myocardial ischemia gene therapy.