Control of three-dimensional substrate stiffness to manipulate mesenchymal stem cell fate toward neuronal or glial lineages

The unlimited self-renewal and multipotency of stem cells provide great potential for applications in tissue engineering and regenerative medicine. The differentiation of stem cells can be induced by multiple factors including physical, chemical and biological cues. The fate of stem cells can be manipulated by deliberately controlling the interaction between stem cells and their microenvironment. The purpose of this study is to investigate the change in matrix stiffness under the influence of neurogenic differentiation of human mesenchymal stem cells (hMSCs). In this study, three-dimensional (3-D) porous scaffolds were synthesized by type I collagen (Col) and hyaluronic acid (HA). The elastic modulus of the 3-D substrates was modified by adjusting the concentration of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent. The mechanical properties of Col–HA scaffolds were evaluated and the induction and characterization of hMSC differentiation toward neural lineages on substrates with different stiffnesses were studied. Using EDC of different concentrations for crosslinking, the stiffness of the matrices can be controlled in the range of 1–10 kPa for soft to stiff substrates, respectively. The results showed that MSCs were likely to differentiate into neuronal lineage in substrate at 1 kPa, while they transformed into glial cells in matrix at 10 kPa. The morphology and proliferation behavior of hMSCs responded to the different stiffnesses of substrates. Using this modifiable matrix, we can investigate the relationship between stem cell behavior and substrate mechanical properties in extracellular matrix-based biomimetic 3-D scaffolds. A substrate with controllable stiffness capable of inducing hMSCs specifically toward neuronal differentiation may be very useful as a tissue-engineered construct or substitute for delivering hMSCs into the brain and spinal cord.     

Matrix Stiffness Modulates Mesenchymal Stem Cell Sensitivity to Geometric Asymmetry Signals

Human stem cells hold significant potential for the treatment of various diseases. However, their use as a therapy is hampered because of limited understanding of the mechanisms by which they respond to environmental stimuli. Efforts to understand extracellular biophysical cues have demonstrated the critical roles of geometrical and mechanical signals in determining the fate of stem cells. The goal of this study was to explore the interplay between cell polarity and matrix stiffness in stem cell lineage specification. We hypothesize that confining cells to asymmetric extracellular matrix islands will impart polarity at a single-cell level and will interact with mechanical signals to define the lineage of stem cells. To test these hypotheses, we employed microcontact printing to create patterned symmetric and asymmetric hydrogel islands of soft and hard surface stiffness. Human mesenchymal stem cells (hMSCs) were confined to these islands at the single-cell level and given the ability to differentiate along adipogenic or osteogenic routes. Our results demonstrated that cell polarity defines the lineage specification of hMSCs only on islands with low stiffness. Insight gained from this study provides a rational basis for designing stem cell cultures to enhance tissue engineering and regenerative medicine strategies.     

The effect of time-dependent deformation of viscoelastic hydrogels on myogenic induction and Rac1 activity in mesenchymal stem cells

Cell behaviours within tissues are influenced by a broad array of physical and biochemical microenvironmental factors. Whilst ‘stiffness’ is a recognised physical property of substrates and tissue microenvironments that influences many cellular behaviours, tissues and their extracellular matrices are not purely rigid but ‘viscoelastic’ materials, composed of both rigid-like (elastic) and dissipative (viscous) elements. This viscoelasticity results in materials displaying increased deformation with time under the imposition of a defined force or stress, a phenomenon referred to as time-dependent deformation or ‘creep’. Previously, we compared the behaviour of human mesenchymal stem cells (hMSCs) on hydrogels tailored to have a constant stiffness, but to display varying levels of creep in response to an applied force. Using polyacrylamide as a model material, we showed that on high-creep hydrogels (HCHs), hMSCs displayed increased proliferation, spread area and differentiation towards multiple lineages, compared to their purely stiff analogue, with a particular propensity for differentiation towards a smooth muscle cell (SMC) lineage. In this present study, we investigate the mechanisms behind this phenomenon and show that hMSCs adhered to HCHs have increased expression of SMC induction factors, including soluble factors, ECM proteins and the cell–cell adhesion molecule, N-Cadherin. Further, we identify a key role for Rac1 signalling in mediating this increased N-Cadherin expression. Using a real-time Rac1-FRET biosensor, we confirm increased Rac1 activation on HCHs, an observation that is further supported functionally by observed increases in motility and lamellipodial protrusion rates of hMSCs. Increased Rac1 activity in hMSCs on HCHs provides underlying mechanisms for enhanced commitment towards a SMC lineage and the compensatory increase in spread area (isotonic tension) after a creep-induced loss of cytoskeletal tension on viscoelastic substrates, in contrast to previous studies that have consistently demonstrated up-regulation of RhoA activity with increasing substrate stiffness. Tuning substrate viscoelasticity to introduce varying levels of creep thus equips the biomaterial scientist or engineer with a new tool with which to tune and direct stem cell outcomes.     

Differentiation of human bone marrow stromal cells into neural-like cells induced by sodium ferulate in vitro

Human marrow stromal cells （hMSCs） are multipotential stem cells, capable of differentiating into bone, cartilage, fat and muscle. Several recent reports demonstrated that hMSCs have been also differentiated into neural cells. However, only a few reported inducers are applicable for clinical use. This work is to explore the effects of sodium ferulate （SF） on differentiation of hMSCs into neural cells in vitro. We found that hMSCs could be induced to the cells with typical neural morphology when cultured with SF. The cells express neural proteins, such as nestin, neuron-specific enolase （NSE） and glial fibrillary acidic protein （GFAP）. About 30% of the hMSC-derived cells expressed nestin when cultured with SF for 3 h, but no expression was detected after 24 h. The percentages of positive cells for NSE or GFAP were about 67% and 39% separately at 6 h, and reached the plateau phage after treatment with SF for 3 days. The data suggest that SF can induce hMSCs to differentiate into neural-like cells in vitro. Cellular ＆ Molecular Immunology. 2005;2（3）：225-229.     

Fast and Efficient Electric‐Triggered Self‐Healing Shape Memory of CNTs@rGO Enhanced PCLPLA Copolymer

Shape memory and self‐healing stimuli‐responsive polymeric materials have aroused extensive research interests due to their special functionality. In this work, a kind of novel and fast electric‐triggered self‐healing shape memory polymeric composite, based on polycaprolactone‐polylactic acid (PCLPLA) copolymer and reduced graphene oxide with embedded carbon nanotubes (CNTs@rGO), are prepared via a facile method. Interestingly, the CNTs@rGO‐PCLPLA composites show improved mechanical properties, excellent electrical conductivity, and could recover their original shape within 60 s by applying 1.42 V mm^?1 electric field. Meanwhile, the proposed networks can be self‐heal rapidly and efficiently under electrothermal effect. These offer for the new material promising practical applications in aerospace, artificial skins, and electronic sensors.     

Poly(N-isopropylacrylamide-co-acrylic acid) nanogels for tracing and delivering genes to human mesenchymal stem cells

Drugs, proteins, and cells can be macro- and micro-encapsulated by unique materials that respond to specific stimuli. The phases and hydrophobic interactions of these materials are reversibly altered by environmental stimuli such as pH and temperature. These changes can lead to self-assembly of the materials, which enables controlled drug release and safe gene delivery into cells and tissues. The fate of stem cells delivered by such methods is of great interest. The formation of transgenic tissues requires genes to be delivered safely into stem cells. A cell tracing vehicle and a gene delivery carrier were simultaneously introduced into human mesenchymal stem cells (hMSCs). A thermo-sensitive hydrogel, poly(N-isopropylacrylamide-co-acrylic acid) (p(NiPAAm-co-AAc)), was created to generate self-assembled nanoparticles with nanogel characteristics. Hydrophobic interactions mediated the binding of the carboxyl group on the outside of p(NiPAAm-co-AAc) with the amine group of iron oxide. Nanogels carrying iron oxide and a fluorescent dye were complexed with specific genes. These nanogels could be internalized by hMSCs, and the transplantation of these cells into mice was monitored by in vivo imaging. Self-assembled p(NiPAAm-co-dAAc) nanogels complexed with green fluorescent protein were highly expressed in hMSCs and are a potential material for gene delivery.     

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.     

Evaluation of Mechanical Properties of Human Mesenchymal Stem Cells During Differentiation to Smooth Muscle Cells

Human mesenchymal stem cells (hMSCs) are multipotent cells appropriate for a variety of tissue engineering and cell therapy applications. Mechanical properties of hMSCs during differentiation are associated with their particular metabolic activity and regulate cell function due to alternations in cytoskeleton and structural elements. The objective of this study is to evaluate elastic and viscoelastic properties of hMSCs during long term cultivation in control and transforming growth factor-β₁ treatment groups using micropipette aspiration technique. The mean Young’s modulus (E) of the control samples remained nearly unchanged during 6 days of cultivation, but that of the test samples showed an initial reduction compared to its relevant control sample after 2 days of treatment by biological growth factor, followed by a significant rise after 4 and 6 days. The viscoelastic creep tests showed that both instantaneous and equilibrium moduli significantly increased with the treatment time and reached to maximum values of 622.9 ± 114.2 and 144.3 ± 11.6 Pa at the sixth day, respectively, while increase in apparent viscosity was not statistically significant. Such change of mechanical properties of hMSCs during specific lineage commitment contributes to regenerative medicine as well as stem-cell-based therapy in which biophysical signals regulate stem cell fate.     

Long-term serial passage and neuronal differentiation capability of human bone marrow mesenchymal stem cells

The development of methods to induce differentiation of human mesenchymal stem cells (hMSCs) has opened the possibility of using these cells in regenerative or reparative therapies. However, the low frequency of hMSCs in tissue means it is often necessary to expand these cells extensively in vitro. In this study, we evaluated the effects of long-term serial passage on the characteristics of bone marrow-derived hMSC populations. In addition, we examined the effect on subsequent hMSC neural differentiation ability, which has not been reported earlier. The hMSC population examined was found to maintain a stable phenotype during the first 6-8 passages of culture as assessed by proliferative ability, morphological appearance, and surface antigen, gene and protein expression, and also expressed pluripotency and neural lineage markers constitutively in the undifferentiated state. Long-term subcultivation neither resulted in spontaneous neural differentiation nor compromised the ability of hMSCs to develop toward an early neuronal fate. In addition, the transformation elicited in hMSC cultures in response to cytokine-based neuronal differentiation was examined by live cell microscopy. We demonstrated, for the first time, that the observed changes result from active and dynamic processes involving outgrowth and motility of cellular extensions, processes entirely distinct from the rapid epiphenomena of cytotoxicity and cytoskeleton disruption generated by chemical induction methods. Cytokine-induced differentiation of hMSCs was also associated with upregulation of early neural gene and protein expression. These findings support the neuronal differentiation capability of hMSCs, although further investigation is required to confirm the ability to attain a mature neuronal phenotype.     

Gadolinium-chelate nanoparticle entrapped human mesenchymal stem cell via photochemical internalization for cancer diagnosis

To improve the gadolinium (Gd) internalization efficiency in stem cells, gadolinium-chelate nanoparticles were prepared from a pullulan derivative (pullulan-deoxycholic acid (DOCA)-diethylene triamine pentaacetic (DTPA)-Gd conjugate; PDDG) and then the PDDG was entrapped into human mesenchymal stem cells (hMSCs) by the photochemical-internalization (PCI) method for cancer diagnosis via the cancer homing property of hMSCs. The internalization efficiency of Gd in hMSCs was significantly increased to 98 ± 4 pg Gd/cell from 32 ± 2 pg Gd/cell via the PCI method. Moreover, the Gd-entrapped hMSCs revealed a low exocytosis ratio of gadolinium-chelate nanoparticles during cell division in vitro and a high cellular labeling efficiency for at least 21 days in vivo. The cancer-targeting and diagnosis effect of the Gd-entrapped hMSCs were confirmed in a small CT26 tumor-bearing mice model. The stem cells detected an early tumor (∼3 mm³) within 2 h using 4.7-T MR and optical imaging. The results demonstrated that the PCI-mediated internalization of Gd-incorporated nanoparticles into hMSCs is a promising protocol for efficient cell labeling and tracking.     

Electroactive porous tubular scaffolds with degradability and non-cytotoxicity for neural tissue regeneration

Electroactive degradable porous tubular scaffolds were fabricated from the blends of polycaprolactone and a hyperbranched degradable conducting copolymer at different feed ratios by a solution-casting/salt-leaching method. Scaning electron microscopy (SEM) and microcomputed tomography tests indicated that these scaffolds had homogeneously distributed interconnected pores on the cross-section and surface. The electrical conductivity of films with the same composition as the scaffolds was between 3.4 × 10⁻⁶ and 3.1 × 10⁻⁷ S cm⁻¹, depending on the ratio of hyperbranched degradable conducting copolymer to polycaprolactone. A hydrophilic surface with a contact angle of water about 30° was achieved by doping the films with (±)-10-camphorsulfonic acid. The mechanical properties of the films were investigated by tensile tests, and the morphology of the films was studied by SEM. The scaffolds were subjected to the WST test (a cell proliferation and cytotoxicity assay using water-soluble tetrazolium salts) with HaCaT keratinocyte cells, and the results show that these scaffolds are non-cytotoxic. These degradable electroactive tubular scaffolds are good candidates for neural tissue engineering application.     

Direct laser machining-induced topographic pattern promotes up-regulation of myogenic markers in human mesenchymal stem cells

The engineering of tissue is preferably done with stem cells, which can be differentiated into the tissue of interest using biochemical or physical cues. While much effort has been focused on using biological factors to regulate stem cell differentiation, recently interest in the contribution of physical factors has increased. In this work, three-dimensional (3-D) microchannels with topographic micropatterns were fabricated by femtosecond laser machining on a biodegradable polymer (poly(L-lactide-co-ε-caprolactone)) substrate. Two substrates with narrow and wide channels respectively were created. Human mesenchymal stem cells (hMSCs) were cultured on the scaffolds for cell proliferation and cellular organization. Gene expression and the immunostaining of myogenic and neurogenic markers were studied. Both scaffolds improved the cell alignment along the channels as compared to the control group. Microfilaments within hMSCs were more significantly aligned and elongated on the narrower microchannels. The gene expression study revealed significant up-regulation of several hallmark markers associated with myogenesis for hMSCs cultured on the scaffold with narrow microchannels, while osteogenic and neurogenic markers were down-regulated or remained similar to the control at day 14. Immunostaining of myogen- and neurogen-specific differentiation markers were used to further confirm the specific differentiation towards a myogenic lineage. This study demonstrates that femtosecond laser machining is a versatile tool for generating controllable 3-D microchannels with topographic features that can be used to induce specific myogenic differentiation of hMSCs in vitro, even in the absence of biological factors.     

Long term human reconstitution and immune aging in NOD-Rag (−)-γ chain (−) mice

Aging of the human immune system is characterized by a gradual loss of immune function and a skewing of hematopoiesis toward the myeloid lineage, a reduction in the lymphocytic lineage, and progressive increases in senescent memory T cells at the expense of naïve T cells. Both the innate and the adaptive branches of the immune system are affected, including neutrophils, macrophages, dendritic cells and lymphocytes. Mice, the most common research model, although inexpensive, do not necessarily reflect the human immune system in terms of its interaction with infectious agents of human origin or environmental factors. This study analyzed whether a human immune system contained within the NOD-Rag (−)-γ chain (−) mouse model could realistically be used to evaluate the development and therapy of aging-related diseases. To that end lightly irradiated NOD-Rag (−)-γ chain (−) mice were injected intra-hepatically on day 1 of life with purified cord blood-derived CD34⁺ stem and progenitor cells. Multiple mice were constructed from each cord blood donor. Mice were analyzed quarterly for age-related changes in the hematopoietic and immune systems, and followed for periods up to 18–24 months post-transplant. Flow cytometric analyses were performed for hematopoietic and immune reconstitution. It was observed that NOD-Rag (−)-γ chain (−) mice could be “humanized” long-term using cord blood stem cells, and that some evidence of immune aging occurred during the life of the mice.     

Quantitative biomarkers of stem cell differentiation based on intrinsic two-photon excited fluorescence

We present the use of two-photon excited fluorescence (TPEF) as a noninvasive means to monitor differentiation of human mesenchymal stem cells (hMSCs) into an adipogenic pathway relying entirely on endogenous sources of contrast. Specifically, we demonstrate that TPEF can be used to reveal quantitative differences in the biochemical status and the shape of differentiating and nondifferentiating stem cells in two-dimensional (2-D) cultures. We find that even in simple 2-D cultures, not all cells are undergoing differentiation at the same rate. Last, such noninvasive approaches may also ultimately allow for determination of the lineage toward which the cells are differentiating (e.g., fat versus bone). Thus, intrinsic TPEF imaging provides quantitative morphological and biochemical biomarkers associated with stem cell differentiation and could serve as an important enabling technology in tissue engineering applications.     

人骨髓间充质干细胞体外内皮细胞诱导分化的研究(英)

目的：探讨人骨髓间充质干细胞hMSCs(human mesenchymal stem cells,hMSCs)体外内皮分化基础及其诱导条件。方法：〖HTSS〗采用密度分离与贴壁筛选结合法分离hMSCs，体外联合应用生长因子VEGF165和不同细胞外基质纤维连接蛋白(FN)与Ⅰ型胶原(Col)，对其进行内皮诱导分化。通过免疫细胞化学、细胞化学、流式细胞分析法及透射电镜对其分化后细胞进行鉴定。结果：hMSCs表达早期内皮分化标志之一KDR；PAS反应阳性及超微结构显示，hMSCs胞浆内含有大量糖原形成糖原池，分化后细胞胞浆外质内糖原明显减少或消失，暗示细胞发生分化；细胞诱导后CE34、β1整合素和KDR表达均增强。结论：诱导后细胞为过渡细胞群类型(transit population,TP)，可向内皮前体细胞(endothelial progenitor cells,EPC)EPC方向分化。推测由此类型细胞再分化为内皮细胞(endothelial cells,ECs)，即hMSCs→TP→EPC→ECs。     

Fates of Fe3O4 and Fe3O4@SiO2 nanoparticles in human mesenchymal stem cells assessed by synchrotron radiation-based techniques

Superparamagnetic iron oxide nanoparticles (SPIOs) have been widely used as the magnetic resonance imaging (MRI) contrast agent in biomedical studies and clinical applications, with special interest recently in in vivo stem cell tracking. However, a full understanding of the fate of SPIOs in cells has not been achieved yet, which is particularly important for stem cells since any change of the microenvironment may disturb their propagation and differentiation behaviors. Herein, synchrotron radiation-based X-ray fluorescence (XRF) in combination with X-ray absorption spectroscopy (XAS) were used to in situ reveal the fate of Fe₃O₄ and Fe₃O₄@SiO₂ NPs in human mesenchymal stem cells (hMSCs), in which the dynamic changes of their distribution and chemical speciation were precisely determined. The XAS analysis evidences that Fe₃O₄ NPs cultured with hMSCs are quite stable and almost keep their initial chemical form up to 14 days, which is contradictory to the previous report that Fe₃O₄ NPs were unstable in cell labeling assessed by using a simplified lysosomal model system. Coating with a SiO₂ shell, Fe₃O₄@SiO₂ NPs present higher stability in hMSCs without detectable changes of their chemical form. In addition, XRF analysis demonstrates that Fe₃O₄@SiO₂ NPs can label hMSCs in a high efficiency manner and are solely distributed in cytoplasm during cell proliferation, making it an ideal probe for in vivo stem cell tracking. These findings with the help of synchrotron radiation-based XAS and XRF improve our understanding of the fate of SPIOs administered to hMSCs and will help the future design of SPIOs for safe and efficient stem cells tracking.     

Effects of Human Fibroblast-Derived Extracellular Matrix on Mesenchymal Stem Cells

Stem cell fate is largely determined by the microenvironment called niche. The extracellular matrix (ECM), as a key component in the niche, is responsible for maintaining structural stability and regulating cell proliferation, differentiation, migration and other cellular activities. Each tissue has a unique ECM composition for its needs. Here we investigated the effect of a bioengineered human dermal fibroblast-derived ECM (hECM) on the regulation of human mesenchymal stem cell (hMSC) proliferation and multilineage differentiation. Human MSCs were maintained on hECM for two passages followed by the analysis of mRNA expression levels of potency- and lineage-specific markers to determine the capacity of MSC stemness and differentiation, respectively. Mesenchymal stem cells pre-cultured with or without hECM were then induced and analyzed for osteogenesis, adipogenesis and chondrogenesis. Our results showed that compared to MSCs maintained on control culture plates without hECM coating, cells on hECM-coated plates proliferated more rapidly with a higher percentage of cells in S phase of the cell cycle, resulting in an increase in the CD90⁺/CD105⁺/CD73⁺/CD45^? subpopulation. In addition, hECM downregulated osteogenesis and adipogenesis of hMSCs but significantly upregulated chondrogenesis with increased production of collagen type 2. In sum, our findings suggest that hECM may be used to culture hMSCs for the application of cartilage tissue engineering.