Calcineurin initiates smooth muscle differentiation in neural crest stem cells

The process of vascular smooth muscle cell (vSMC) differentiation is critical to embryonic angiogenesis. However, despite its importance, the vSMC differentiation program remains largely undefined. Murine gene disruption studies have identified several gene products that are necessary for vSMC differentiation, but these methodologies cannot establish whether or not a factor is sufficient to initiate the differentiation program. A gain-of-function system consisting of normal vSMC progenitor cells would serve as a useful complement to whole animal loss-of-function studies. We use such a system here, namely freshly isolated rat neural crest stem cells (NCSCs), to show that activation of the calcineurin signaling pathway is sufficient to drive these cells toward a smooth muscle fate. In addition, we present data suggesting that transforming growth factor (TGF)-beta1, which also causes NCSCs to differentiate into smooth muscle, activates calcineurin signaling in NCSCs, leading to a model in which activation of calcineurin signaling is the mechanism by which TGF-beta1 causes SMC differentiation in these cells.     

Matrix stiffness modulates the differentiation of neural crest stem cells in vivo

Stem cells are often transplanted with scaffolds for tissue regeneration; however, how the mechanical property of a scaffold modulates stem cell fate in vivo is not well understood. Here we investigated how matrix stiffness modulates stem cell differentiation in a model of vascular graft transplantation. Multipotent neural crest stem cells (NCSCs) were differentiated from induced pluripotent stem cells, embedded in the hydrogel on the outer surface of nanofibrous polymer grafts, and implanted into rat carotid arteries by anastomosis. After 3 months, NCSCs differentiated into smooth muscle cells (SMCs) near the outer surface of the polymer grafts; in contrast, NCSCs differentiated into glial cells in the most part of the hydrogel. Atomic force microscopy demonstrated a stiffer matrix near the polymer surface but much lower stiffness away from the polymer graft. Consistently, in vitro studies confirmed that stiff surface induced SMC genes whereas soft surface induced glial genes. These results suggest that the scaffold’s mechanical properties play an important role in directing stem cell differentiation in vivo, which has important implications in biomaterials design for stem cell delivery and tissue engineering.     

Regulation of vascular smooth muscle cells and mesenchymal stem cells by mechanical strain

Vascular smooth muscle cells (SMCs) populate in the media of the blood vessel, and play an important role in the control of vasoactivity and the remodeling of the vessel wall. Blood vessels are constantly subjected to hemodynamic stresses, and the pulsatile nature of the blood flow results in a cyclic mechanical strain in the vessel walls. Accumulating evidence in the past two decades indicates that mechanical strain regulates vascular SMC phenotype, function and matrix remodeling. Bone marrow mesenchymal stem cell (MSC) is a potential cell source for vascular regeneration therapy, and may be used to generate SMCs to construct tissue-engineered vascular grafts for blood vessel replacements. In this review, we will focus on the effects of mechanical strain on SMCs and MSCs, e.g., cell phenotype, cell morphology, cytoskeleton organization, gene expression, signal transduction and receptor activation. We will compare the responses of SMCs and MSCs to equiaxial strain, uniaxial strain and mechanical strain in three-dimensional culture. Understanding the hemodynamic regulation of SMC and MSC functions will provide a basis for the development of new vascular therapies and for the construction of tissue-engineered vascular grafts.     

Prokineticin-1 modulates proliferation and differentiation of enteric neural crest cells

Prokineticins (Prok-1 and Prok-2) belong to a newly identified AVIT protein family. They are involved in variety of activities in various tissues, including smooth muscle contraction of the gastrointestinal tract and promoting proliferation of endothelial cells derived from adrenal gland. Importantly, they also act as the survival factors to modulate growth and survival of neurons and hematopoietic stem cells. In this study we demonstrated that Prok-1 (but not Prok-2) protein is expressed in the mucosa and mesenchyme of the mouse embryonic gut during enteric nervous system development. Its receptor, PK-R1 is expressed in the enteric neural crest cells (NCCs). To elucidate the physiological role(s) of Prok-1 in NCCs, we isolated the NCCs from the mouse embryonic gut (E11.5) and cultured them in the form of neurospheres. In an in vitro NCC culture, Prok-1 was able to activate both Akt and MAPK pathways and induce the proliferation and differentiation (but not migration) of NCCs via PK-R1. Knock-down of PK-R1 using siRNA resulted in a complete abolishment of Prok-1 induced proliferation. Taken together, it is the first report demonstrating that Prok-1 acts as a gut mucosa/mesenchyme-derived factor and maintains proliferation and differentiation of enteric NCCs.     

27nt-miRNA对间充质干细胞向血管平滑肌细胞分化的影响

为探讨27nt-miRNA对间充质干细胞向血管平滑肌细胞分化影响,构建27nt-miRNA过表达、反义序列Anti-27nt-miRNA以及阴性对照的表达质粒,慢病毒包装后分别转染人脐带间充质干细胞(hUCMSC),加入Ⅳ型胶原诱导hUCMSC定向分化为血管平滑肌细胞。四唑盐(MTT)比色法检测分化后细胞活力,免疫细胞化学染色法检测分化后细胞SM22α(兔抗平滑肌22α,smooth muscle 22α)的表达,Western印迹法和RT-PCR检测分化后细胞内的SMA (兔抗平滑肌肌动蛋白,smooth muscle actin) mRNA、SM 22α mRNA及其蛋白质表达情况。经检测,27nt-miRNA过表达分化组与阴性对照组相比,细胞活力下降20.48%(P0.05),SMA mRNA、SM22α mRNA及其蛋白质表达量明显升高(P0.05);而Anti-27nt-miRNA分化组细胞活力上升了18.07%(P0.05),SMA mRNA、SM22α mRNA及其蛋白质表达量下降(P0.05)。综上所述,27nt-miRNA能够促进间充质干细胞向血管平滑肌细胞分化,并且抑制分化后的细胞活力。     

Effects of micropatterned curvature on the motility and mechanical properties of airway smooth muscle cells

Geometric features such as size and shape of the microenvironment are known to alter cell behaviors such as growth, differentiation, apoptosis, and migration. Little is known, however, about the effect of curvature on cell behaviors despite that many cells reside in curved space of tubular organs such as the bronchial airways. To address this question, we fabricated micropatterned strips that mimic airway walls with varying curvature. Then, we cultured airway smooth muscle cells (ASMCs) on these strips and investigated the cells’ motility and mechanical properties using time-lapse imaging microscopy and optical magnetic twisting cytometry (OMTC). We found that both motility and mechanical properties of the ASMCs were influenced by the curvature. In particular, when the curvature increased from 0 to 1/150 μm⁻¹, the velocity of cell migration first decreased (0–1/750 μm⁻¹), and then increased (1/750–1/150 μm⁻¹). In contrast, the cell stiffness increased and then decreased. Thus, at the intermediate curvature (1/750 μm⁻¹) the ASMCs were the least motile, but most stiff. The contractility instead decreased consistently as the curvature increased. The level of F-actin, and vinculin expression within the ASMCs appeared to correlate with the contractility and motility, respectively, in relation to the curvature. These results may provide valuable insights to understanding the heterogeneity of airway constrictions in asthma as well as the developing and functioning of other tubular organs and tissue engineering.     

Bcl-XL modulates the differentiation of immortalized human neural stem cells

Understanding basic processes of human neural stem cell (hNSC) biology and differentiation is crucial for the development of cell replacement therapies. Bcl-X(L) has been reported to enhance dopaminergic neuron generation from hNSCs and mouse embryonic stem cells. In this work, we wanted to study, at the cellular level, the effects that Bcl-X(L) may exert on cell death during differentiation of hNSCs, and also on cell fate decisions and differentiation. To this end, we have used both v-myc immortalized (hNS1 cell line) and non-immortalized neurosphere cultures of hNSCs. In culture, using different experimental settings, we have consistently found that Bcl-X(L) enhances neuron generation while precluding glia generation. These effects do not arise from a glia-to-neuron shift (changes in fate decisions taken by precursors) or by only cell death counteraction, but, rather, data point to Bcl-X(L) increasing proliferation of neuronal progenitors, and inhibiting the differentiation of glial precursors. In vivo, after transplantation into the aged rat striatum, Bcl-X(L) overexpressing hNS1 cells generated more neurons and less glia than the control ones, confirming the results obtained in vitro. These results indicate an action of Bcl-X(L) modulating hNSCs differentiation, and may be thus important for the future development of cell therapy strategies for the diseased mammalian brain.     

Effects of transforming growth factor-beta 1 and ascorbic acid on differentiation of human bone-marrow-derived mesenchymal stem cells into smooth muscle cell lineage

Bone-marrow-derived mesenchymal stem cells (MSCs) can differentiate into a variety of cell types including smooth muscle cells (SMCs). We have attempted to demonstrate that, following treatment with transforming growth factor-beta 1 (TGF-beta1) and ascorbic acid (AA), human bone-marrow-derived MSCs differentiate into the SMC lineage for use in tissue engineering. Quantitative polymerase chain reaction for SMC-specific gene (alpha smooth muscle actin, h1-calponin, and SM22alpha) expression was performed on MSCs, which were cultured with various concentrations of TGF-beta1 or AA. TGF-beta1 had a tendency to up-regulate the expression of SMC-specific genes in a dose-dependent manner. The expression of SM22alpha was significantly up-regulated by 30 muM AA. We also investigated the additive effect of TGF-beta1 and AA for differentiation into SMCs and compared this effect with that of other factors including platelet-derived growth factor BB (PDGF-BB). In addition to SMC-specific gene expression, SMC-specific proteins increased by two to four times when TGF-beta1 and AA were used together compared with their administration alone. PDGF did not increase the expression of SMC-specific markers. MSCs cultured with TGF-beta1 and AA did not differentiate into osteoblasts and adipocytes. These results suggest that a combination of TGF-beta1 and AA is useful for the differentiation of MSCs into SMCs for use in tissue engineering.     

Fibronectin promotes differentiation of neural crest progenitors endowed with smooth muscle cell potential

The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effect was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells.     

Cyclic strain induces mouse embryonic stem cell differentiation into vascular smooth muscle cells by activating PDGF receptor beta

Embryonic stem (ES) cells are exposed to fluid-mechanical forces, such as cyclic strain and shear stress, during the process of embryonic development but much remains to be elucidated concerning the role of fluid-mechanical forces in ES cell differentiation. Here, we show that cyclic strain induces vascular smooth muscle cell (VSMC) differentiation in murine ES cells. Flk-1-positive (Flk-1+) ES cells seeded on flexible silicone membranes were subjected to controlled levels of cyclic strain and examined for changes in cell proliferation and expression of various cell lineage markers. When exposed to cyclic strain (4-12% strain, 1 Hz, 24 h), the Flk-1+ ES cells significantly increased in cell number and became oriented perpendicular to the direction of strain. There were dose-dependent increases in the VSMC markers smooth muscle alpha-actin and smooth muscle-myosin heavy chain at both the protein and gene expression level in response to cyclic strain, whereas expression of the vascular endothelial cell marker Flk-1 decreased, and there were no changes in the other endothelial cell markers (Flt-1, VE-cadherin, and platelet endothelial cell adhesion molecule 1), the blood cell marker CD3, or the epithelial marker keratin. The PDGF receptor beta (PDGFR beta) kinase inhibitor AG-1296 completely blocked the cyclic strain-induced increase in cell number and VSMC marker expression. Cyclic strain immediately caused phosphorylation of PDGFR beta in a dose-dependent manner, but neutralizing antibody against PDGF-BB did not block the PDGFR beta phosphorylation. These results suggest that cyclic strain activates PDGFR beta in a ligand-independent manner and that the activation plays a critical role in VSMC differentiation from Flk-1+ ES cells.     

Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells

Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest-related disorders.     

Uniaxial strain upregulates matrix-degrading enzymes produced by human vascular smooth muscle cells

Arteries remodel in response to environmental changes. We investigated whether mechanical strain modulates production of matrix metalloproteinase (MMP)-2 and -9 by cultured vascular smooth muscle cells (SMC). MMP-2 and MMP-9 expression were tested using human saphenous vein SMC cultured on silicone membranes at rest or subjected to physiological levels (5%) of stationary or cyclical (1 Hz) uniaxial strain. Compared with control, stationary strain significantly increased MMP-2 mRNA levels at all time points, whereas cyclic strain decreased it after 48 h. Both secreted and cell-associated pro-MMP-2 levels were increased by stationary strain at all times (P < 0.01), whereas cyclic strain decreased secreted levels after 48 h (P < 0.02). MMP-9 mRNA levels and pro-MMP-9 protein were increased after 48 h of stationary stretch (P < 0.01) compared with both no strain and cyclic strain. Our study indicates that vascular SMC show a selective response to different types of strain. We suggest that local increases in stationary mechanical strain resulting from stenting, hypertension, or atherosclerosis may lead to enhanced matrix degradation by SMC.     

The stem cells of the neural crest

In the vertebrate embryo, the neurectodermal neural crest cells (NCC) have remarkably broad potencies, giving rise, after a migratory phase, to neurons and glial cells in the peripheral nervous system, and to skin melanocytes, being all designated here as “neural” derivatives. NC-derived cells also include non-neural, “mesenchymal” cell types like chondrocytes and bone cells, myofibroblasts and adipocytes, which largely contribute to the head structures in amniotes. Similar to the blood cell system, the NC is therefore a valuable model to investigate the mechanisms of cell lineage diversification in vertebrates. Whether NCC are endowed with multiple differentiation potentials or if, conversely, they are a mosaic of different committed cells is an important ongoing issue to understand the ontogeny of NC derivatives in normal development and pathological conditions. Here we focus on recent findings that established the presence in the early migratory NC of the avian embryo, of a multipotent progenitor endowed with both mesenchymal and neural differentiation capacities. This “mesenchymal-neural” clonogenic cell lies upstream of all the other NC progenitors known so far and shows increased frequency when single cell cultures are treated with the Sonic Hedgehog signaling molecule. These findings are discussed in the context of the broad potentials of NC stem cells recently evidenced in certain adult mammalian tissues.     

Endoglin is required for myogenic differentiation potential of neural crest stem cells

Genetic studies show that TGFbeta signaling is essential for vascular development, although the mechanism through which this pathway operates is incompletely understood. Here we demonstrate that the TGFbeta auxiliary coreceptor endoglin (eng, CD105) is expressed in a subset of neural crest stem cells (NCSCs) in vivo and is required for their myogenic differentiation. Overexpression of endoglin in the neural crest caused pericardial hemorrhaging, correlating with altered vascular smooth muscle cell investment in the walls of major vessels and upregulation of smooth muscle alpha-actin protein levels. Clonogenic differentiation assay of NCSCs derived from neural tube explants demonstrated that only NCSC expressing high levels of endoglin (NCSC(CD105+)) had myogenic differentiation potential. Furthermore, myogenic potential was deficient in NCSCs obtained from endoglin null embryos. Expression of endoglin in NCSCs declined with age, coinciding with a reduction in both smooth muscle differentiation potential and TGFbeta1 responsiveness. These findings demonstrate a cell autonomous role for endoglin in smooth muscle cell specification contributing to vascular integrity.     

Directed differentiation of embryonic P19 cells and neural stem cells into neural lineage on conducting PEDOT–PEG and ITO glass substrates

Differentiation of pluripotent and lineage restricted stem cells such as neural stem cells (NSCs) was studied on conducting substrates of various nature without perturbation of the genome with exogenous genetic material or chemical stimuli. Primary mouse adult neural stem cells (NSCs) and P19 pluripotent embryonal (P19 EC) carcinoma cells were used. Expression levels of neuronal markers β-III-tubulin and neurofilament were evaluated by immunochemistry and flow cytometry. It was shown that the ability of the substrate to induce differentiation directly correlated with its conductivity. Conducting substrates (conducting oxides or doped π-conjugated organic polymers) with different morphology, structure, and conductivity mechanisms all promoted differentiation of NSC and P19 cells into neuronal lineage to a similar degree without use of additional factors such as poly-l-ornithine coating or retinoic acid, as verified by their morphology and upregulation of the neuronal markers but not astrocyte marker GFAP. However, substrates with low conductance below ca. 10^?4 S cm^?2 did not show this ability. Morphology of differentiating cells was visualized by atomic force microscopy. NSCs cells increased β-III-tubulin expression by 95% and P19 cells by over 30%. Our results suggest that the substrate conductivity is a key factor governing the cell fate. Differentiation of P19 cells into neuronal lineage on conducting substrates was attributed to downregualtion of Akt signaling pathway and increase in expression of dual oxidase 1 (DUOX 1).     

Engineering micropatterned surfaces to modulate the function of vascular stem cells

Multipotent vascular stem cells have been implicated in vascular disease and in tissue remodeling post therapeutic intervention. Hyper-proliferation and calcified extracellular matrix deposition of VSC cause blood vessel narrowing and plaque hardening thereby increasing the risk of myocardial infarct. In this study, to optimize the surface design of vascular implants, we determined whether micropatterned polymer surfaces can modulate VSC differentiation and calcified matrix deposition. Undifferentiated rat VSC were cultured on microgrooved surfaces of varied groove widths, and on micropost surfaces. 10 μm microgrooved surfaces elongated VSC and decreased cell proliferation. However, microgrooved surfaces did not attenuate calcified extracellular matrix deposition by VSC cultured in osteogenic media conditions. In contrast, VSC cultured on micropost surfaces assumed a dendritic morphology, were significantly less proliferative, and deposited minimal calcified extracellular matrix. These results have significant implications for optimizing the design of cardiovascular implant surfaces.     

Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells

To isolate mouse neural crest stem cells, we have generated a rat monoclonal antibody to murine neurotrophin receptor (p75). We have immortalized p75⁺ murine neural crest cells by expression of v-myc, and have isolated several clonal cell lines. These lines can be maintained in an undifferentiated state, or induced to differentiate by changing the culture conditions. One of these cell lines, MONC-1, is capable of generating peripheral neurons, glia, and melanocytic cells. Importantly, most individual MONC-1 cells are multipotent when analyzed at clonal density. The neurons that differentiate under standard conditions have an autonomic-like phenotype, but under different conditions can express markers of other peripheral neuronal lineages. These lines therefore exhibit a similar differentiation potential as their normal counterparts. Furthermore, they can be genetically modified or generated from mice of different genetic backgrounds, providing a useful tool for molecular studies of neural crest development. © 1997 John Wiley & Sons, Inc. J Neuroblol 32 : 722–746, 1997     

Cooperation of myocardin and Smad2 in inducing differentiation of mesenchymal stem cells into smooth muscle cells

Several reports demonstrated that mesenchymal stem cells (MSCs) might differentiate into smooth muscle cells (SMCs) in vitro and in vivo. It has been shown that myocardin protein is a strong inducer of smooth muscle genes and MSCs can differentiate into SMCs in response to transforming growth factor-β (TGF-β). However, the relationship or link between myocardin and TGF-β3-induced MSC differentiation has not been fully elucidated. Here, we demonstrated that both myocardin and TGF-β3 were able to induce differentiation of rat bone marrow-derived MSCs toward smooth-muscle-like cell types, as evidenced by increasing expression of SMC-specific genes. Of note, myocardin cooperated with Smad2 to synergistically activate SM22α promoter and significantly enhance the expression of SM22α. Report assays with site-direct mutation analysis of SM22α promoter demonstrated that myocardin and Smad2 coactivated SM22α promoter mainly depending on CArG box and less on smad binding elements (SBE) sites as well. These findings reveal the cooperation of myocardin and Smad2 in process of MSC differentiation into SMCs.     

Identification of early neurogenic cells in the neural crest lineage.

Pluripotent neural crest cells are restricted progressively during development. The sequence of restrictions and the time(s) in early development at which such restrictions are imposed on crest-derived cells are largely unknown. We have used a human autoantibody (Anti-Hu) to characterize neurogenic populations of avian neural crest-derived cells. Anti-Hu binds specifically to neurons and neuroendocrine cells in older (greater than E4) quail embryos. Early in development, Anti-Hu also binds a subpopulation of neural crest-derived cells that lack neuronal morphology and do not express other neuronal traits. These cells may represent a putative neurogenic precursor subpopulation within the early crest cell lineage. To test this hypothesis, we have characterized Anti-Hu immunoreactivity within crest-derived populations known to have, or to lack, the ability to give rise to new neurons. We report that the presence of Anti-Hu+ nonneuronal cells is correlated with the neurogenic ability of a given cell population. Moreover, Anti-Hu+ nonneuronal cells are transient and appear to be replaced by Anti-Hu+ neuronal cells. We conclude that Anti-Hu is a very early indicator of neurogenesis among crest-derived cells and that Anti-Hu+ nonneuronal cells are either neurogenic precursors or immature neurons.