Stimulation of adipogenesis of adult adipose-derived stem cells using substrates that mimic the stiffness of adipose tissue

Biochemical and biomechanical extracellular matrix (ECM) cues have recently been shown to play a role in stimulating stem cell differentiation towards several lineages, though how they combine to induce adipogenesis has been less well studied. The objective of this study was to recapitulate both the ECM composition and mechanical properties of adipose tissue in vitro to stimulate adipogenesis of human adipose-derived stem cells (ASCs) in the absence of exogenous adipogenic growth factors and small molecules. Adipose specific ECM biochemical cues have been previously shown to influence adipogenic differentiation; however, the ability of biomechanical cues to promote adipogenesis has been less defined. Decellularized human lipoaspirate was used to functionalize polyacrylamide gels of varying stiffness to allow the cells to interact with adipose-specific ECM components. Culturing ASCs on gels that mimicked the native stiffness of adipose tissue (2 kPa) significantly upregulated adipogenic markers, in the absence of exogenous adipogenic growth factors and small molecules. As substrate stiffness increased, the cells became more spread, lost their rounded morphology, and failed to upregulate adipogenic markers. Together these data imply that as with other lineages, mechanical cues are capable of regulating adipogenesis in ASCs.     

Functional Modulation of ES-Derived Hepatocyte Lineage Cells via Substrate Compliance Alteration

Pluripotent embryonic stem cells represent a promising renewable cell source to generate a variety of differentiated cell types including hepatocyte lineage cells, and may ultimately be incorporated into extracorporeal bioartificial liver devices and cell replacement therapies. Recently, we and others have utilized sodium butyrate to directly differentiate hepatocyte-like cells from murine embryonic stem cells cultured in a monolayer configuration. However, to incorporate stem cell technology into clinical and pharmaceutical applications, and hopefully increase the therapeutic potential of these differentiated cells for liver disease treatment, a major challenge remains in sustaining differentiated functions for an extended period of time in their secondary culture environment. In the present work, we have investigated the use of polyacrylamide hydrogels with defined mechanical compliances as a cell culture platform for improving and/or stabilizing functions of these hepatocyte-like cells. Several functional assays, e.g., urea secretion, intracellular albumin content, and albumin secretion, were performed to characterize hepatic functions of cells on polyacrylamide gels with stiffnesses of 5, 46.6, and 230 kPa. In conjunction with the mechanical and cell morphological characterization, we showed that hepatic functions of sodium butyrate differentiated cells were sustained and further enhanced on compliant substrates. This study promises to offer insights into regulating stem cell differentiation via mechanical stimuli, and assist us with designing a variety of dynamic culture systems for applications in tissue and cellular engineering.     

软基质力学特性对滑膜间充质干细胞向软骨细胞分化的干预作用

背景：课题组前期研究表明,较软的培养基质对大鼠骨髓间充质干细胞的形态及细胞骨架有明显的影响。 目的：探讨不同弹性模量的聚丙烯酰胺凝胶软基质对人滑膜间充质干细胞向软骨细胞分化的影响。 方法：无菌条件下获取骨关节炎患者滑膜组织,有限稀释法获得原代人滑膜间充质干细胞,流式细胞术进行细胞表面标记物鉴定,多向诱导分化实验进行功能鉴定。用丙烯酰胺和甲叉双丙烯酰胺制备0.4,6,30 kPa 3个弹性模量的聚丙烯酰胺凝胶软基质作为培养人滑膜间充质干细胞的基底,在转化生长因子β1存在的情况下分别培养7 d和14 d,RT-PCR方法检测软骨形成相关基因COL2A1、CRTAC1 mRNA的表达,以6孔细胞培养板作为对照组。 结果与结论：在不同弹性模量上生长的人滑膜间充质干细胞表现出不同的细胞形态;软基质的弹性模量及培养时间对人滑膜间充质干细胞成软骨基因COL2A1、CRTAC1的表达有交互作用：7 d时,CRTAC1 mRNA在6 kPa弹性模量聚丙烯酰胺凝胶上表达量最高(F=44.350,P=0.000);7 d时,COL2A1 mRNA在0.4 kPa弹性模量聚丙烯酰胺软基质上的表达量最高(F=6.384,P=0.005)。较低弹性模量的聚丙烯酰胺凝胶软基质比常规细胞培养板更具有促进滑膜间充质干细胞向软骨细胞分化的作用。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程      

Evaluation of Lateral Mechanical Tension in Thin-Film Tissue Constructs

Fibroblast-populated collagen matrices provide a simplified tissue model for wound healing and development processes. A technology (CELLDRUM Technology) evaluating lateral mechanical tension in fibroblast-populated collagen matrices (tissue constructs) with a thickness of 1 mm was introduced. Defined mechanical boundary conditions together with the known number and orientation of the cells revealed precise data on the average tension exerted by a single cell. Circular cell-populated collagen gels were manufactured inside the CELLDRUM on top of a flexible membrane. The collagen matrix was then excited by a sound pulse. The resulting resonance oscillation was monitored by a laser-based deflection sensor and frequency and damping were analyzed giving information on mechanical properties of the tissue construct. Several evaluation experiments were performed. Calf serum enhanced contractile forces of fibroblasts dose dependently. After the gels were treated with cytochalasin D for 24 h, the cell forces were reduced by 42% of control. The remaining tension was attributed to the extracellular matrix remodeling occurring during cell growth and to other cytoskeletal structures like microtubules and intermediate filaments. We also found that only after a few hours of culture fibroblast-seeded collagen gels began developing significant mechanical tension. A mechanical tension profile of proliferating fibroblasts in collagen gels over culture time was obtained.     

心脏干细胞分离培养及其增殖与迁移的机制

文章快速阅读：文题释义： 干细胞niche：在心脏干细胞的自我更新、生长增殖、多向分化和持续存活等方面需要依赖特定的微环境,即干细胞niche。干细胞niche对干细胞的作用是通过多种因素起作用的,例如干细胞间的相互作用、干细胞与邻近分化细胞的相互作用、干细胞与细胞因子、生长因子及黏附因子的相互作用等,干细胞niche调控干细胞自我更新、多向分化是一个复杂的网络。 心肌重构：是由一系列复杂的分子和细胞机制导致心肌结构、功能和表型的变化,这些变化包括心肌细胞肥大、细胞凋亡、胚胎基因和蛋白质的再表达、心肌细胞外基质量和组成的变化。 摘要 背景：体外环境下进行心脏干细胞培养的过程中,生长微环境可能会对细胞的的增殖等产生一定的影响。 目的：在体外环境下培养大鼠心脏干细胞,探讨心脏干细胞增殖、迁移的可能机制。 方法：纳入SD大鼠10只,获得心肌组织块进行原代和传代培养。收集第3代心脏干细胞进行免疫荧光染色,进行干细胞鉴定,检测干细胞生长因子受体(c-kit)和CD45、CD90的表达。收集培养后的组织块,随机分为2 份,一份用多聚甲醛进行固定,常规石蜡包埋后切片,进行苏木精-伊红染色、Masson染色以及细胞凋亡检测;另一份在培养基中添加EdU标记增殖细胞,采用免疫荧光染色检测c-kit阳性细胞以及基质金属蛋白酶2,9及转化生长因子β1等表达。 结果与结论：①细胞鉴定结果：心肌组织培养7-10 d后有明亮的圆形细胞长出,细胞贴壁后呈梭状,生长、增殖能力旺盛。免疫荧光染色可观察到大量细胞呈c-kit、CD45阳性表达,CD90阴性表达;②细胞凋亡情况：培养的组织块切片后染色见大量新生细胞生长的同时伴随心肌细胞的凋亡;③免疫荧光染色结果：经EdU标记后阳性细胞分布于心肌间隙,基质金属蛋白酶2,9及转化生长因子β1少量表达,但周围心肌组织上述3因子表达明显增多。④结果表明,心肌组织体外培养可获取心脏干细胞,伴随细胞增殖和迁移,其机制与基质金属蛋白酶2,9及转化生长因子β1的表达有关。  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程 ORCID: 0000-0002-7743-0450(侯波)     

Pluripotent stem cell-derived cardiac tissue patch with advanced structure and function

Recent advances in pluripotent stem cell research have provided investigators with potent sources of cardiogenic cells. However, tissue engineering methodologies to assemble cardiac progenitors into aligned, 3-dimensional (3D) myocardial tissues capable of physiologically relevant electrical conduction and force generation are lacking. In this study, we introduced 3D cell alignment cues in a fibrin-based hydrogel matrix to engineer highly functional cardiac tissues from genetically purified mouse embryonic stem cell-derived cardiomyocytes (CMs) and cardiovascular progenitors (CVPs). Procedures for CM and CVP derivation, purification, and functional differentiation in monolayer cultures were first optimized to yield robust intercellular coupling and maximize velocity of action potential propagation. A versatile soft-lithography technique was then applied to reproducibly fabricate engineered cardiac tissues with controllable size and 3D architecture. While purified CMs assembled into a functional 3D syncytium only when supplemented with supporting non-myocytes, purified CVPs differentiated into cardiomyocytes, smooth muscle, and endothelial cells, and autonomously supported the formation of functional cardiac tissues. After a total culture time similar to period of mouse embryonic development (21 days), the engineered cardiac tissues exhibited unprecedented levels of 3D organization and functional differentiation characteristic of native neonatal myocardium, including: 1) dense, uniformly aligned, highly differentiated and electromechanically coupled cardiomyocytes, 2) rapid action potential conduction with velocities between 22 and 25 cm/s, and 3) significant contractile forces of up to 2 mN. These results represent an important advancement in stem cell-based cardiac tissue engineering and provide the foundation for exploiting the exciting progress in pluripotent stem cell research in the future tissue engineering therapies for heart disease.     

Pluripotent stem cell-engineered cell sheets reassembled with defined cardiovascular populations ameliorate reduction in infarct heart function through cardiomyocyte-mediated neovascularization

Although stem cell therapy is a promising strategy for cardiac restoration, the heterogeneity of transplanted cells has been hampering the precise understanding of the cellular and molecular mechanisms. Previously, we established a cardiovascular cell differentiation system from mouse pluripotent stem cells, in which cardiomyocytes (CMs), endothelial cells (ECs), and mural cells (MCs) can be systematically induced and purified. Combining this with cell sheet technology, we generated cardiac tissue sheets reassembled with defined cardiovascular populations. Here, we show the potentials and mechanisms of cardiac tissue sheet transplantation in cardiac function after myocardial infarction (MI). Transplantation of the cardiac tissue sheet to a rat MI model showed significant and sustained improvement of systolic function accompanied by neovascularization. Reduction of the infarct wall thinning and fibrotic length indicated the attenuation of left ventricular remodeling. Cell tracing with species-specific fluorescent in situ hybridization after transplantation revealed a relatively early loss of transplanted cells and an increase in endogenous neovascularization in the proximity of the graft, suggesting an indirect angiogenic effect of cardiac tissue sheets rather than direct CM contributions. We prospectively dissected the functional mechanisms with cell type-controlled sheet analyses. Sheet CMs were the main source of vascular endothelial growth factor. Transplantation of sheets lacking CMs resulted in the disappearance of neovascularization and subsequent functional improvement, indicating that the beneficial effects of the sheet were achieved by sheet CMs. ECs and MCs enhanced the sheet functions and structural integration. Supplying CMs to ischemic regions with cellular interaction could be a strategic key in future cardiac cell therapy.     

Generation of cardiac spheres from primate pluripotent stem cells in a small molecule-based 3D system

Pluripotent stem cell (PSC) usage in heart regenerative medicine requires producing enriched cardiomyocytes (CMs) with mature phenotypes in a defined medium. However, current methods are typically performed in 2D environments that produce immature CMs. Here we report a simple, growth factor-free 3D culture system to rapidly and efficiently generate 85.07  ±  1.8% of spontaneously contractile cardiac spheres (scCDSs) using 3D-cultured human and monkey PSC-spheres. Along with small molecule-based 3D induction, this protocol produces CDSs of up to 95.7% CMs at a yield of up to 237 CMs for every input pluripotent cell, is effective for human and monkey PSCs, and maintains 81.03  ±  12.43% of CDSs in spontaneous contractibility for over three months. These CDSs displayed CM ultrastructure, calcium transient, appropriate pharmacological responses and CM gene expression profiles specific for maturity. Furthermore, 3D-derived CMs displayed more mature phenotypes than those from a parallel 2D-culture. The system is compatible to large-scaly produce CMs for disease study, cell therapy and pharmaceutics screening.     

The Current Status of iPS Cells in Cardiac Research and Their Potential for Tissue Engineering and Regenerative Medicine

The recent availability of human cardiomyocytes derived from induced pluripotent stem (iPS) cells opens new opportunities to build in vitro models of cardiac disease, screening for new drugs, and patient-specific cardiac therapy. Notably, the use of iPS cells enables studies in the wide pool of genotypes and phenotypes. We describe progress in reprogramming of induced pluripotent stem (iPS) cells towards the cardiac lineage/differentiation. The focus is on challenges of cardiac disease modeling using iPS cells and their potential to produce safe, effective and affordable therapies/applications with the emphasis of cardiac tissue engineering. We also discuss implications of human iPS cells to biological research and some of the future needs.     

Cartilaginous constructs using primary chondrocytes from continuous expansion culture seeded in dense collagen gels

Cell-based therapies such as autologous chondrocyte implantation require in vitro cell expansion. However, standard culture techniques require cell passaging, leading to dedifferentiation into a fibroblast-like cell type. Primary chondrocytes grown on continuously expanding culture dishes (CE culture) limits passaging and protects against dedifferentiation. The authors tested whether CE culture chondrocytes were advantageous for producing mechanically competent cartilage matrix when three-dimensionally seeded in dense collagen gels. Primary chondrocytes, grown either in CE culture or passaged twice on static silicone dishes (SS culture; comparable to standard methods), were seeded in dense collagen gels and cultured for 3 weeks in the absence of exogenous chondrogenic growth factors. Compared with gels seeded with SS culture chondrocytes, CE chondrocyte-seeded gels had significantly higher chondrogenic gene expression after 2 and 3 weeks in culture, correlating with significantly higher aggrecan and type II collagen protein accumulation. There was no obvious difference in glycosaminoglycan content from either culture condition, yet CE chondrocyte-seeded gels were significantly thicker and had a significantly higher dynamic compressive modulus than SS chondrocyte-seeded gels after 3 weeks. Chondrocytes grown in CE culture and seeded in dense collagen gels produce more cartilaginous matrix with superior mechanical properties, making them more suitable than SS cultured cells for tissue engineering applications.     

Engineered heart slices for electrophysiological and contractile studies

A major consideration in the design of engineered cardiac tissues for the faithful representation of physiological behavior is the recapitulation of the complex topography and biochemistry of native tissue. In this study we present engineered heart slices (EHS), which consist of neonatal rat ventricular cells (NRVCs) seeded onto thin slices of decellularized cardiac tissue that retain important aspects of native extracellular matrix (ECM). To form EHS, rat or pig ventricular tissue was sectioned into 300 μm-thick, 5 to 16 mm-diameter disks, which were subsequently decellularized using detergents, spread on coverslips, and seeded with NRVCs. The organized fiber structure of the ECM remained after decellularization and promoted cell elongation and alignment, resulting in an anisotropic, functional tissue that could be electrically paced. Contraction decreased at higher pacing rates, and optical mapping revealed electrical conduction that was anisotropic with a ratio of approximately 2.0, rate-dependent shortening of the action potential and slowing of conduction, and slowing of conduction by the sodium channel blocker lidocaine. Reentrant arrhythmias could also be pace-induced and terminated. EHS constitute an attractive in vitro cardiac tissue in which cardiac cells are cultured on thin slices of decellularized cardiac ECM that provide important biochemical, structural, and mechanical cues absent in traditional cell cultures.     

The effect of vitronectin on the differentiation of embryonic stem cells in a 3D culture system

While stem cell niches in vivo are complex three-dimensional (3D) microenvironments, the relationship between the dimensionality of the niche to its function is unknown. We have created a 3D microenvironment through electrospinning to study the impact of geometry and different extracellular proteins on the development of cardiac progenitor cells (Flk-1⁺) from resident stem cells and their differentiation into functional cardiovascular cells. We have investigated the effect of collagen IV, fibronectin, laminin and vitronectin on the adhesion and proliferation of murine ES cells as well as the effects of these proteins on the number of Flk-1⁺ cells cultured in 2D conditions compared to 3D system in a feeder free condition. We found that the number of Flk-1⁺ cells was significantly higher in 3D scaffolds coated with laminin or vitronectin compared to colIV-coated scaffolds. Our results show the importance of defined culture systems in vitro for studying the guided differentiation of pluripotent embryonic stem cells in the field of cardiovascular tissue engineering and regenerative medicine.     

Lateral boundary mechanosensing by adherent cells in a collagen gel system

Cell adhesion responses to in-depth physical properties such as substrate roughness and topography are well described but little is known about the influence of lateral physical cues such as tissue boundaries on the function of adherent cells. Accordingly, we developed a model system to examine remote cell sensing of lateral boundaries. The model employs floating thin collagen gels supported by rigid grids of varying widths. The dynamics, lengths, and numbers of cell extensions were regulated by grid opening size, which in turn determined the distance of cells from rigid physical boundaries. In smaller grids (200 μm and 500 μm wide), cell-induced deformation fields extended to, and were resisted by, the grid boundaries. However, in larger grids (1700 μm wide), the deformation field did not extend to the grid boundaries, which strongly affected the mean length and number of cell extensions (∼60% reduction). The generation of cell extensions in collagen gels required expression of the β1 integrin, focal adhesion kinase and actomyosin activity. We conclude that the presence of physical boundaries interrupts the process of cell-mediated collagen compaction and fiber alignment in the collagen matrix and enhances the formation of cell extensions. This new cell culture platform provides a geometry that more closely approximates the native basement membrane and will help to elucidate the roles of cell extensions and lateral mechanosensing on extracellular matrix remodeling by invasion and degradation.     

Osteoblasts derived from induced pluripotent stem cells form calcified structures in scaffolds both in vitro and in vivo

Reprogramming somatic cells into an ESC-like state, or induced pluripotent stem (iPS) cells, has emerged as a promising new venue for customized cell therapies. In this study, we performed directed differentiation to assess the ability of murine iPS cells to differentiate into bone, cartilage, and fat in vitro and to maintain an osteoblast phenotype on a scaffold in vitro and in vivo. Embryoid bodies derived from murine iPS cells were cultured in differentiation medium for 8–12 weeks. Differentiation was assessed by lineage-specific morphology, gene expression, histological stain, and immunostaining to detect matrix deposition. After 12 weeks of expansion, iPS-derived osteoblasts were seeded in a gelfoam matrix followed by subcutaneous implantation in syngenic imprinting control region (ICR) mice. Implants were harvested at 12 weeks, histological analyses of cell and mineral and matrix content were performed. Differentiation of iPS cells into mesenchymal lineages of bone, cartilage, and fat was confirmed by morphology and expression of lineage-specific genes. Isolated implants of iPS cell-derived osteoblasts expressed matrices characteristic of bone, including osteocalcin and bone sialoprotein. Implants were also stained with alizarin red and von Kossa, demonstrating mineralization and persistence of an osteoblast phenotype. Recruitment of vasculature and microvascularization of the implant was also detected. Taken together, these data demonstrate functional osteoblast differentiation from iPS cells both in vitro and in vivo and reveal a source of cells, which merit evaluation for their potential uses in orthopedic medicine and understanding of molecular mechanisms of orthopedic disease.     

Multifunctional and stable bone mimic proteinaceous matrix for bone tissue engineering

Biomaterial surface design with biomimetic proteins holds great promise for successful regeneration of tissues including bone. Here we report a novel proteinaceous hybrid matrix mimicking bone extracellular matrix that has multifunctional capacity to promote stem cell adhesion and osteogenesis with excellent stability. Osteocalcin-fibronectin fusion protein holding collagen binding domain was networked with fibrillar collagen, featuring bone extracellular matrix mimic, to provide multifunctional and structurally-stable biomatrices. The hybrid protein, integrated homogeneously with collagen fibrillar networks, preserved structural stability over a month. Biological efficacy of the hybrid matrix was proven onto tethered surface of biopolymer porous scaffolds. Mesenchymal stem cells quickly anchored to the hybrid matrix, forming focal adhesions, and substantially conformed to cytoskeletal extensions, benefited from the fibronectin adhesive domains. Cells achieved high proliferative capacity to reach confluence rapidly and switched to a mature and osteogenic phenotype more effectively, resulting in greater osteogenic matrix syntheses and mineralization, driven by the engineered osteocalcin. The hybrid biomimetic matrix significantly improved in vivo bone formation in calvarial defects over 6 weeks. Based on the series of stimulated biological responses in vitro and in vivo the novel hybrid proteinaceous composition will be potentially useful as stem cell interfacing matrices for osteogenesis and bone regeneration.     

Geometric guidance of integrin mediated traction stress during stem cell differentiation

Cells sense and transduce the chemical and mechanical properties of their microenvironment through cell surface integrin receptors. Traction stress exerted by cells on the extracellular matrix mediates focal adhesion stabilization and regulation of the cytoskeleton for directing biological activity. Understanding how stem cells integrate biomaterials properties through focal adhesions during differentiation is important for the design of soft materials for regenerative medicine. In this paper we use micropatterned hydrogels containing fluorescent beads to explore force transmission through integrins from single mesenchymal stem cells (MSCs) during differentiation. When cultured on polyacrylamide gels, MSCs will express markers associated with osteogenesis and myogenesis in a stiffness dependent manner. The shape of single cells and the composition of tethered matrix protein both influence the magnitude of traction stress applied and the resultant differentiation outcome. We show how geometry guides the spatial positioning of focal adhesions to maximize interaction with the matrix, and uncover a relationship between αvβ3, α5β1 and mechanochemical regulation of osteogenesis.     

Paper-based bioactive scaffolds for stem cell-mediated bone tissue engineering

Bioactive, functional scaffolds are required to improve the regenerative potential of stem cells for tissue reconstruction and functional recovery of damaged tissues. Here, we report a paper-based bioactive scaffold platform for stem cell culture and transplantation for bone reconstruction. The paper scaffolds are surface-engineered by an initiated chemical vapor deposition process for serial coating of a water-repellent and cell-adhesive polymer film, which ensures the long-term stability in cell culture medium and induces efficient cell attachment. The prepared paper scaffolds are compatible with general stem cell culture and manipulation techniques. An optimal paper type is found to provide structural, physical, and mechanical cues to enhance the osteogenic differentiation of human adipose-derived stem cells (hADSCs). A bioactive paper scaffold significantly enhances in vivo bone regeneration of hADSCs in a critical-sized calvarial bone defect. Stacking the paper scaffolds with osteogenically differentiated hADSCs and human endothelial cells resulted in vascularized bone formation in vivo. Our study suggests that paper possesses great potential as a bioactive, functional, and cost-effective scaffold platform for stem cell-mediated bone tissue engineering. To the best of our knowledge, this is the first study reporting the feasibility of a paper material for stem cell application to repair tissue defects.