线粒体的力学生物学研究进展

线粒体是高度动态的细胞器,不仅为细胞提供能量和物质基础,同时参与调控细胞的增殖、迁移、分化和凋亡等。细胞命运受来自微环境的力学信号调节,近年来的研究表明,力学因素对细胞的能量代谢具有调控作用,线粒体作为一个力学感受器和枢纽连接力学和代谢来调控细胞的命运。深入理解力学微环境和线粒体代谢的关系,为促进组织再生和疾病治疗提供有力的指导。本文主要介绍线粒体力学生物学研究进展,并探讨其在组织再生和疾病治疗中的潜在应用。     

The effects of interactive mechanical and biochemical niche signaling on osteogenic differentiation of adipose-derived stem cells using combinatorial hydrogels

Stem cells reside in a multi-factorial environment containing biochemical and mechanical signals. Changing biochemical signals in most scaffolds often leads to simultaneous changes in mechanical properties, which makes it difficult to elucidate the complex interplay between niche cues. Combinatorial studies on cell–material interactions have emerged as a tool to facilitate analyses of stem cell responses to various niche cues, but most studies to date have been performed on two-dimensional environments. Here we developed three-dimensional combinatorial hydrogels with independent control of biochemical and mechanical properties to facilitate analysis of interactive biochemical and mechanical signaling on adipose-derived stem cell osteogenesis in three dimensions. Our results suggest that scaffold biochemical and mechanical signals synergize only at specific combinations to promote bone differentiation. Leading compositions were identified to have intermediate stiffness (～55 kPa) and low concentration of fibronectin (10 μg ml^?1), which led to an increase in osteocalcin gene expression of over 130-fold. Our results suggest that scaffolds with independently tunable niche cues could provide a powerful tool for conducting mechanistic studies to decipher how complex niche cues regulate stem cell fate in three dimensions, and facilitate rapid identification of optimal niche cues that promote desirable cellular processes or tissue regeneration.     

Regulation of the fate of human mesenchymal stem cells by mechanical and stereo-topographical cues provided by silicon nanowires

Extracellular stimuli imposed on stem cells enable efficient initiation of mechanotransductive signaling to regulate stem cell fates; however, how such physical cues conferred by the stereo-topographical matrix govern the fate of stem cells still remains unknown. The purpose of this study is to delineate the effects of stereotopography and its various relevant physical properties on the fate regulation of human mesenchymal stem cells (hMSCs). Stereo-topographical silicon nanowires (SiNWs) that were precisely controlled with respect to their various dimensions and their growth orientation were used in this study. hMSCs cultured on stereo SiNWs of different lengths in the absence of biochemical osteogenic induction cues displayed a spherical and less-elongated morphology and showed an approximately 10% loss of cell viability compared to those grown on two-dimensional (2-D) flat Si. Moreover, osteogenic gene expression of COL1A1 and Runx2 in hMSCs cultured on the shortest SiNWs was significantly higher than those grown on the longer SiNWs and 2-D flat Si. hMSCs grown on shorter SiNWs also demonstrated higher expression levels for F-actin, phosphorylated focal adhesion kinase (pFAK), vinculin and alpha 2 integrin. Stereo-topographical cues provided by SiNWs are able to regulate osteogenic differentiation of hMSCs via cytoskeleton remodeling and this is correlated with the differential expression of alpha 2/beta 1 integrin heterodimers and the focal adhesion molecules pFAK and vinculin. The findings in this study provide insights in terms of the design of stereo-topographical structures for use in tissue engineering, bone regeneration and relevant medical applications.     

Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage

Neural stem cells (NSCs) yield both neuronal and glial progeny, but their differentiation potential toward multiple region-specific neuron types remains remarkably poor. In contrast, embryonic stem cell (ESC) progeny readily yield region-specific neuronal fates in response to appropriate developmental signals. Here we demonstrate prospective and clonal isolation of neural rosette cells (termed R-NSCs), a novel NSC type with broad differentiation potential toward CNS and PNS fates and capable of in vivo engraftment. R-NSCs can be derived from human and mouse ESCs or from neural plate stage embryos. While R-NSCs express markers classically associated with NSC fate, we identified a set of genes that specifically mark the R-NSC state. Maintenance of R-NSCs is promoted by activation of SHH and Notch pathways. In the absence of these signals, R-NSCs rapidly lose rosette organization and progress to a more restricted NSC stage. We propose that R-NSCs represent the first characterized NSC stage capable of responding to patterning cues that direct differentiation toward region-specific neuronal fates. In addition, the R-NSC-specific genetic markers presented here offer new tools for harnessing the differentiation potential of human ESCs.     

A new perspective for stem-cell mechanobiology: biomechanical control of stem-cell behavior and fate

Biomechanics is known to play an important role in cell metabolism. Cell phenotype, tissue-specific functions, and fate critically depend on the extracellular mechanical environment. The mechanical properties of the cell itself, such as cytoskeleton elasticity, membrane tension, and adhesion strength, may also play an important role in cell homeostasis and differentiation. Pluripotent bone marrow-derived human mesenchymal stem cells, for example, can be differentiated into many tissue-specific lineages. While cellular biomechanical properties are significantly altered during stem-cell specification to a particular phenotype, the complexity of events associated with transformation of these precursor cells leaves many questions unanswered about morphological, structural, proteomic, and functional changes in differentiating stem cells. A thorough understanding of stem-cell behavior would allow the development of more effective approaches to the expansion of stem cells in vitro and the regulation of their commitment to a specific phenotype. Control of cell behaviors might be feasible through manipulation of the cellular biomechanical properties using various external physical stimuli, including electric fields, mechanical stimuli, and genetic manipulation of the expression of particular genes. Biomechanical regulation of stem-cell differentiation can greatly minimize the number of chemicals and growth factors that would otherwise be required for composite tissue engineering. Determination and the appropriate use of the known physicochemical cues will facilitate current research effort toward designing and engineering functional tissue constructs.     

Directed growth and differentiation of stem cells towards neural cell fates using soluble and surface-mediated cues

Stem and progenitor cells are helping researchers understand the complex process of mammalian development and also show great promise in treating diseases that are unresponsive to standard therapies. The potential for embryonic stem cells to differentiate into any cell in the body is their great benefit but avoiding co-culture with animal cells and efficiently narrowing cell fate to a single cell type remains challenging. Adult progenitor cells have a more restricted cell fate, but have the potential for use in autologous cell therapies and avoid the ethical issues surrounding the derivation of embryonic stem cell lines. While progress is encouraging, there is much work to be done in directing cells to specific lineages before stem and progenitor cells can be commonly used in clinical settings. This review discusses current techniques used for investigation of the growth and differentiation of stem and progenitor cells, with a focus on neural cell fates.     

Guidance of stem cell fate on 2D patterned surfaces

Stem cells possess unique abilities as they can renew themselves for extended periods of time and have the capacity to differentiate into a variety of lineages. They hold promise for treating a plethora of diseases ranging from musculoskeletal defects to myocardial infarction and to neural disorders. Understanding how to control the fate decision of these cells to self-renew or differentiate is paramount in stem cell tissue engineering. Recently, significant progress has been made in guiding stem cell differentiation in?vitro, and we are beginning to understand the complex interplay of factors that control their fate. Here, we highlight the recent approaches for guidance of stem cells through patterning of surfaces at the micro- and nanoscale. Particular attention is given to chemical patterning of substrates with adhesion ligands and physical patterning with a variety of topographical features. These surface-mediated biochemical and mechanical cues have proven influential in altering a wide range of stem cell phenotypes. This approach to guide or ultimately control stem cells by surface patterning has enormous potential implications in cell therapies and regenerative medicine.     

Mechanical control of stem cell differentiation

Numerous studies have focused on identifying the chemical and biological factors that govern the differentiation of stem cells; however, recent research has shown that mechanical cues may play an equally important role. Mechanical forces such as shear stresses and tensile loads, as well as the rigidity and topography of the extracellular matrix were shown to induce significant changes in the morphology and fate of stem cells. We survey experimental studies that focused on the response of stem cells to mechanical and geometrical properties of their environment and discuss the mechanical mechanisms that accompany their response including the remodeling of the cytoskeleton and determination of cell and nucleus size and shape.     

Bioprinting of growth factors onto aligned sub-micron fibrous scaffolds for simultaneous control of cell differentiation and alignment

The capability to spatially control stem cell orientation and differentiation simultaneously using a combination of geometric cues that mimic structural aspects of native extracellular matrix (ECM) and biochemical cues such as ECM-bound growth factors (GFs) is important for understanding the organization and function of musculoskeletal tissues. Herein, oriented sub-micron fibers, which are morphologically similar to musculoskeletal ECM, were spatially patterned with GFs using an inkjet-based bioprinter to create geometric and biochemical cues that direct musculoskeletal cell alignment and differentiation in vitro in registration with fiber orientation and printed patterns, respectively. Sub-micron polystyrene fibers (diameter ~ 655 nm) were fabricated using a Spinneret-based Tunable Engineered Parameters (STEP) technique and coated with serum or fibrin. The fibers were subsequently patterned with tendon-promoting fibroblast growth factor-2 (FGF-2) or bone-promoting bone morphogenetic protein-2 (BMP-2) prior to seeding with mouse C2C12 myoblasts or C3H10T1/2 mesenchymal fibroblasts. Unprinted regions of STEP fibers showed myocyte differentiation while printed FGF-2 and BMP-2 patterns promoted tenocyte and osteoblast fates, respectively, and inhibited myocyte differentiation. Additionally, cells aligned along the fiber length. Functionalizing oriented sub-micron fibers with printed GFs provides instructive cues to spatially control cell fate and alignment to mimic native tissue organization and may have applications in regenerative medicine.     

The Advancement of Biomaterials in Regulating Stem Cell Fate

Stem cells are well-known to have prominent roles in tissue engineering applications. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can differentiate into every cell type in the body while adult stem cells such as mesenchymal stem cells (MSCs) can be isolated from various sources. Nevertheless, an utmost limitation in harnessing stem cells for tissue engineering is the supply of cells. The advances in biomaterial technology allows the establishment of ex vivo expansion systems to overcome this bottleneck. The progress of various scaffold fabrication could direct stem cell fate decisions including cell proliferation and differentiation into specific lineages in vitro. Stem cell biology and biomaterial technology promote synergistic effect on stem cell-based regenerative therapies. Therefore, understanding the interaction of stem cell and biomaterials would allow the designation of new biomaterials for future clinical therapeutic applications for tissue regeneration. This review focuses mainly on the advances of natural and synthetic biomaterials in regulating stem cell fate decisions. We have also briefly discussed how biological and biophysical properties of biomaterials including wettability, chemical functionality, biodegradability and stiffness play their roles.     

The influence of hydrogel modulus on the proliferation and differentiation of encapsulated neural stem cells

There has been an increasing interest in understanding how the mechanical properties of the microenvironment influence stem cell fate. We describe studies of the proliferation and differentiation of neural stem cells (NSCs) encapsulated within three-dimensional scaffolds – alginate hydrogels – whose elastic moduli were varied over two orders of magnitude. The rate of proliferation of neural stem cells decreased with increase in the modulus of the hydrogels. Moreover, we observed the greatest enhancement in expression of the neuronal marker β-tubulin III within the softest hydrogels, which had an elastic modulus comparable to that of brain tissues. To our knowledge, this work represents the first demonstration of the influence of modulus on NSC differentiation in three-dimensional scaffolds. Three-dimensional scaffolds that control stem cell fate would be broadly useful for applications in regenerative medicine and tissue engineering.     

Directing stem cell fate on hydrogel substrates by controlling cell geometry,matrix mechanics and adhesion ligand composition

There is a dynamic relationship between physical and biochemical signals presented in the stem cell microenvironment to guide cell fate determination. Model systems that modulate cell geometry, substrate stiffness or matrix composition have proved useful in exploring how these signals influence stem cell fate. However, the interplay between these physical and biochemical cues during differentiation remains unclear. Here, we demonstrate a microengineering strategy to vary single cell geometry and the composition of adhesion ligands — on substrates that approximate the mechanical properties of soft tissues — to study adipogenesis and neurogenesis in adherent mesenchymal stem cells. Cells cultured in small circular islands show elevated expression of adipogenesis markers while cells that spread in anisotropic geometries tend to express elevated neurogenic markers. Arraying different combinations of matrix protein in a myriad of 2D and pseudo-3D geometries reveals optimal microenvironments for controlling the differentiation of stem cells to these “soft” lineages without the use of media supplements.     

Stem cell plasticity: learning from hepatogenic differentiation strategies.

Many studies on stem cell plasticity are challenging the concept that stem cells contain an intrinsically predefined, unidirectional differentiation program. This means that the developmental fate of a stem cell is dependent on the general potential of the cell (pre-determined stem cell fate) as well as on microenvironmental cues, such as stimuli from growth factors (stem cell niche). Here, we reviewed reports that examined the hepatocyte differentiation ability of stem cells from two different sources: embryonic stem cells and adult stem cells. All of those stem cells revealed the ability to give rise to hepatocyte-like cells using different induction strategies. However, it is still not clear which of those stem cells would be the best source for hepatocyte replacement or which would be the best protocol. We herein present the current knowledge regarding available protocols and factors used in order to obtain functional hepatocytes from stem cells.     

Stem cell plasticity in mammals and transdetermination in Drosophila: common themes?

Stem cells have been identified in a number of mammalian tissues (e.g. bone marrow, muscle, gut, skin, and neural tissues). Until recently, it was generally believed that the differentiation potential of a mammalian somatic stem cell is restricted to one tissue only, as in the case of hematopoietic stem cells differentiating into hematopoietic cells. In this sense, somatic stem cells are limited in their differentiation potential. Several lines of evidence now challenge the idea of unilateral development. New reports show mammalian somatic stem cells can, in the course of regeneration, repopulate heterologous cell systems and therefore possess a surprisingly broad spectrum of differentiation potential. Thus, mammalian stem cells are apparently capable of fate changes between stem cell systems, although the mechanisms leading to such changes are unclear. Mechanistic models for fate changes have been proposed in Drosophila, specifically for transdetermination of imaginal discs. Imaginal discs of the larva are the primordia of the adult exoskeleton and appendages, for example, legs, and antennae. Transplantation experiments of imaginal discs have shown that discs are determined for their disc identity. Transdetermination in Drosophila refers to cases when, after regenerative cell divisions, imaginal disc cells change from one state of determination to another, initiating a pathway of differentiation leading to structures other than those corresponding to the initial state or determination; for example, an antennal imaginal disc transdetermines to a leg imaginal disc. A fate change is thus possible in both mammalian somatic stem cells and Drosophila imaginal discs following transplantation and subsequent proliferation. Here we summarize and compare observations made in such cases of stem cell and imaginal disc differentiation.     

A review of the effects of the cell environment physicochemical nanoarchitecture on stem cell commitment

Physicochemical features of a cell nanoenvironment exert important influence on stem cell behavior and include the influence of matrix elasticity and topography on differentiation processes. The presence of growth factors such as TGF-β and BMPs on these matrices provides chemical cues and thus plays vital role in directing eventual stem cell fate. Engineering of functional biomimetic scaffolds that present programmed spatio-temporal physical and chemical signals to stem cells holds great promise in stem cell therapy. Progress in this field requires tacit understanding of the mechanistic aspects of cell-environment nanointeractions, so that they can be manipulated and exploited for the design of sophisticated next generation biomaterials. In this review, we report and discuss the evolution of these processes and pathways in the context of matrix adhesion as they might relate to stemness and stem cell differentiation. Super-resolution microscopy and single-molecule methods for in vitro nano-manipulation are helping to identify and characterize the molecules and mechanics of structural transitions within stem cells and matrices. All these advances facilitate research toward understanding of stem cell niche and consequently to developing new class of biomaterials helping the “used biomaterials” for applications in tissue engineering and regenerative medicine.     

Scaffold-based approach to direct stem cell neural and cardiovascular differentiation: an analysis of physical and biochemical effects

Although stem cell therapy holds tremendous promise in tissue regeneration and disease treatment, its full potential may only be realized through the thorough understanding and capability in specifically directing stem cell fate commitment. A scaffold-based approach of imparting physical and biochemical cues appears to be a logical method in reconstructing the complex three-dimensional configuration of stem cell niches. In fact, interest in this area has gained significant momentum over the recent years. This review summarizes and evaluates the recent outcomes of studies associated with a scaffold-based approach to directing and understanding stem cell neural and cardiovascular differentiation.     

Design of tissue engineering scaffolds as delivery devices for mechanical and mechanically modulated signals

New approaches to tissue engineering aim to exploit endogenous strategies such as those occurring in prenatal development and recapitulated during postnatal healing. Defining tissue template specifications to mimic the environment of the condensed mesenchyme during development allows for exploitation of tissue scaffolds as delivery devices for extrinsic cues, including biochemical and mechanical signals, to drive the fate of mesenchymal stem cells seeded within. Although a variety of biochemical signals that modulate stem cell fate have been identified, the mechanical signals conducive to guiding pluripotent cells toward specific lineages are less well characterized. Furthermore, not only is spatial and temporal control of mechanical stimuli to cells challenging, but also tissue template geometries vary with time due to tissue ingrowth and/or scaffold degradation. Hence, a case study was carried out to analyze flow regimes in a testbed scaffold as a first step toward optimizing scaffold architecture. A pressure gradient was applied to produce local (nm-micron) flow fields conducive to migration, adhesion, proliferation, and differentiation of cells seeded within, as well as global flow parameters (micron-mm), including flow velocity and permeability, to enhance directed cell infiltration and augment mass transport. Iterative occlusion of flow channel dimensions was carried out to predict virtually the effect of temporal geometric variation (e.g., due to tissue development and growth) on delivery of local and global mechanical signals. Thereafter, insights from the case study were generalized to present an optimization scheme for future development of scaffolds to be implemented in vitro or in vivo. Although it is likely that manufacture and testing will be required to finalize design specifications, it is expected that the use of the rational design optimization will reduce the number of iterations required to determine final prototype geometries and flow conditions. As the range of mechanical signals conducive to guiding cell fate in situ is further elucidated, these refined design criteria can be integrated into the general optimization rubric, providing a technological platform to exploit nature's endogenous tissue engineering strategies for targeted tissue generation in the lab or the clinic.