Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes

Myocardin is a serum response factor (SRF) coactivator exclusively expressed in cardiomyocytes and smooth muscle cells (SMCs). However, there is highly controversial evidence as to whether myocardin is essential for normal differentiation of these cell types, and there are no data showing whether cardiac or SMC subtypes exhibit differential myocardin requirements during development. Results of the present studies showed the virtual absence of myocardin(-/-) visceral SMCs or ventricular myocytes in chimeric myocardin knockout (KO) mice generated by injection of myocardin(-/-) embryonic stem cells (ESCs) into wild-type (WT; i.e., myocardin(+/+) ESC) blastocysts. In contrast, myocardin(-/-) ESCs readily formed vascular SMC, albeit at a reduced frequency compared with WT ESCs. In addition, myocardin(-/-) ESCs competed equally with WT ESCs in forming atrial myocytes. The ultrastructural features of myocardin(-/-) vascular SMCs and cardiomyocytes were unchanged from their WT counterparts as determined using a unique X-ray microprobe transmission electron microscopic method developed by our laboratory. Myocardin(-/-) ESC-derived SMCs also showed normal contractile properties in an in vitro embryoid body SMC differentiation model, other than impaired thromboxane A2 responsiveness. Together, these results provide novel evidence that myocardin is essential for development of visceral SMCs and ventricular myocytes but is dispensable for development of atrial myocytes and vascular SMCs in the setting of chimeric KO mice. In addition, results suggest that as yet undefined defects in development and/or maturation of ventricular cardiomyocytes may have contributed to early embryonic lethality observed in conventional myocardin KO mice and that observed deficiencies in development of vascular SMC may have been secondary to these defects.     

Towards the maturation and characterization of smooth muscle cells derived from human embryonic stem cells

In this study we demonstrate that CD34(+) cells derived from human embryonic stem cells (hESCs) have higher smooth muscle cell (SMC) potential than CD34(-) cells. We report that from all inductive signals tested, retinoic acid (RA) and platelet derived growth factor (PDGF(BB)) are the most effective agents in guiding the differentiation of CD34(+) cells into smooth muscle progenitor cells (SMPCs) characterized by the expression of SMC genes and proteins, secretion of SMC-related cytokines, contraction in response to depolarization agents and vasoactive peptides and expression of SMC-related genes in a 3D environment. These cells are also characterized by a low organization of the contractile proteins and the contractility response is mediated by Ca(2+), which involves the activation of Rho A/Rho kinase- and Ca(2+)/calmodulin (CaM)/myosin light chain kinase (MLCK)-dependent pathways. We further show that SMPCs obtained from the differentiation of CD34(+) cells with RA, but not with PDGF(BB,) can be maturated in medium supplemented with endothelin-1 showing at the end individualized contractile filaments. Overall the hESC-derived SMCs presented in this work might be an unlimited source of SMCs for tissue engineering and regenerative medicine.     

成体干细胞向类许旺细胞诱导分化研究进展

周围神经损伤修复的黄金标准是自体神经移植,但由于遗留供区部位感觉功能障碍及可供移植神经数量有限,致使该项技术在临床上的应用受到限制,组织工程学的发展为此提供了一种新的解决途径.许旺细胞是周围神经组织工程重要的种子细胞,在神经再生过程中发挥重要作用,移植许旺细胞修复周围神经损伤有广阔的应用前景,但异体移植常面临免疫排斥反应,这些均导致了许旺细胞作为周围神经组织工程的种子细胞在临床应用中受到限制.近年来,随着对具有自我更新和多向分化潜能的间充质干细胞的深入研究,为组织工程化人工神经种子细胞的发展提出新的思路,本文将各种成体干细胞作为许旺细胞的替代细胞的研究进展作一综述.     

Soft substrate maintains stemness and pluripotent stem cell-like phenotype of human embryonic stem cells under defined culture conditions

Cytotechnology - Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the pre-implantation blastocyst. Prior to embryo implantation, the ICM cells are surrounded by...     

Transforming growth factor-beta1 signaling contributes to development of smooth muscle cells from embryonic stem cells

Knockout of transforming growth factor (TGF)-1 or components of its signaling pathway leads to embryonic death in mice due to impaired yolk sac vascular development before significant smooth muscle cell (SMC) maturation occurs. Thus the role of TGF-1 in SMC development remains unclear. Embryonic stem cell (ESC)-derived embryoid bodies (EBs) recapitulate many of the events of early embryonic development and represent a more physiological context in which to study SMC development than most other in vitro systems. The present studies showed induction of the SMC-selective genes smooth muscle -actin (SMA), SM22, myocardin, smoothelin-B, and smooth muscle myosin heavy chain (SMMHC) within a mouse ESC-EB model system. Significantly, SM2, the SMMHC isoform associated with fully differentiated SMCs, was expressed. Importantly, the results showed that aggregates of SMMHC-expressing cells exhibited visible contractile activity, suggesting that all regulatory pathways essential for development of contractile SMCs were functional in this in vitro model system. Inhibition of endogenous TGF- with an adenovirus expressing a soluble truncated TGF- type II receptor attenuated the increase in SMC-selective gene expression in the ESC-EBs, as did an antibody specific for TGF-1. Of interest, the results of small interfering (si)RNA experiments provided evidence for differential TGF--Smad signaling for an early vs. late SMC marker gene in that SMA promoter activity was dependent on both Smad2 and Smad3 whereas SMMHC activity was Smad2 dependent. These results are the first to provide direct evidence that TGF-1 signaling through Smad2 and Smad3 plays an important role in the development of SMCs from totipotential ESCs. embryoid body; Smad     

Differentiation of human embryonic stem cells into smooth muscle cells in adherent monolayer culture

Smooth muscle cell (SMC) plays critical roles in many human diseases, an in vitro system that recapitulates human SMC differentiation would be invaluable for exploring molecular mechanisms leading to the human diseases. We report a directed and highly efficient SMC differentiation system by treating the monolayer-cultivated human embryonic stem cells (hESCs) with all-trans retinoid acid (atRA). When the hESCs were cultivated in differentiation medium containing 10microM RA, more than 93% of the cells expressed SMC-marker genes along with the steadily accumulation of such SMC-specific proteins as SM alpha-actin and SM-MHC. The fully differentiated SMCs were stable in phenotype and capable of contraction. This inducible and highly efficient in vitro human SMC system could be an important resource to study the mechanisms of SMC phenotype determination in human.     

Expression of the dystrophin-glycoprotein complex is a marker for human airway smooth muscle phenotype maturation

Airway smooth muscle (ASM) cells may contribute to asthma pathogenesis through their capacity to switch between a synthetic/proliferative and a contractile phenotype. The multimeric dystrophin-glycoprotein complex (DGC) spans the sarcolemma, linking the actin cytoskeleton and extracellular matrix. The DGC is expressed in smooth muscle tissue, but its functional role is not fully established. We tested whether contractile phenotype maturation of human ASM is associated with accumulation of DGC proteins. We compared subconfluent, serum-fed cultures and confluent cultures subjected to serum deprivation, which express a contractile phenotype. Western blotting confirmed that beta-dystroglycan, beta-, delta-, and epsilon-sarcoglycan, and dystrophin abundance increased six- to eightfold in association with smooth muscle myosin heavy chain (smMHC) and calponin accumulation during 4-day serum deprivation. Immunocytochemistry showed that the accumulation of DGC subunits was specifically localized to a subset of cells that exhibit robust staining for smMHC. Laminin competing peptide (YIGSR, 1 microM) and phosphatidylinositol 3-kinase (PI3K) inhibitors (20 microM LY-294002 or 100 nM wortmannin) abrogated the accumulation of smMHC, calponin, and DGC proteins. These studies demonstrate that the accumulation of DGC is an integral feature for phenotype maturation of human ASM cells. This provides a strong rationale for future studies investigating the role of the DGC in ASM smooth muscle physiology in health and disease.     

Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization

Although isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts were used, a mechanical method of passaging led to better cell growth than passaging by trypsin digestion. We also found that exogenous supplementation with leukemia inhibitory factor maintained the embryonic stem cell-like cells in an undifferentiated state, whereas addition of stem cell factor resulted in their differentiation. Our findings provide an experimental basis for the establishment of an effective culture system for bovine embryonic stem cells.     

Heparin and the phenotype of adult human vascular smooth muscle cells

Summary To study mechanisms controlling growth and phenotype in human vascular smooth muscle cells, we established culture conditions under which these cells proliferate rapidly and achieve life-spans of 50–60 population doublings. In medium containing heparin and heparin-binding growth factors, growth rate and life-span of human vascular smooth muscle cells increased more than 50% relative to cultures with neither supplement, and more than 20% compared to cultures supplemented only with heparin-binding growth factors. In contrast to observations made in rat vascular smooth muscle cells, smooth muscle-specific α-actin in the human cells was expressed only in the presence of heparin and colocalized with β/γ nonmuscle actins in stress fibers, not in adhesion plaques. Heparin, in the presence of heparin-binding growth factors, also caused more than 170% stimulation of tracer glucosamine incorporation into hyaluronic acid and a 7.5-fold increase in hyaluronic acid accumulation. In comparison, total sulfate incorporation into sulfated glycosaminoglycans increased by less than 40%. In light of our previous findings that heparin suppresses collagen gene expression, we conclude that heparin induces human vascular smooth muscle cells exposed to heparin-binding growth factors to remodel their extracellular matrix by altering the relative rates of hyaluronic acid (HA) and collagen synthesis. The resulting hyaluronic-acid-rich, collagen-poor matrix may enhance infiltration of CD44/hyaluronate-receptor-bearing T-lymphocytes and monocytes into the vascular wall, an early event in atherogenesis.     

Integrated membrane protein analysis of mature and embryonic stem cell-derived smooth muscle cells using a novel combination of CyDye/biotin labeling

Cultivated vascular smooth muscle cells (SMCs) were surface-labeled with CyDyes followed by biotinylation. After enrichment on avidin columns, proteins were separated on large format gradient gels by SDS-PAGE. A comparison between CyDye-tagged and non-tagged gel bands revealed a substantial increase of protein identifications from membrane, membrane-associated, and extracellular matrix proteins with a corresponding reduction in co-purified intracellular proteins. Notably the majority of identified proteins were involved in cellular adhesion processes. To demonstrate the quantitative potential of this platform, we performed a comparison between mature and embryonic stem cell-derived smooth muscle cells (esSMCs) and identified the membrane proteins E-cadherin, integrin alpha6, and CD98 (4F2) to be significantly up-regulated in esSMCs suggesting that SMCs derived from embryonic stem cells maintain characteristics of their embryonic stem cell origin. This was subsequently confirmed by RT-PCR: despite expressing a panel of smooth muscle markers (calponin, Sm22, and aortic smooth muscle actin), esSMCs remained positive for markers of stem cell pluripotency (Oct4, Nanog, and Rex1). In summary, we describe a novel strategy for the profiling of cell membrane proteins. The procedure combines DIGE technology with biotin/avidin labeling to discriminate membrane and membrane-associated proteins from intracellular contaminants by fluorescence tagging and permits semiquantitative differential expression analysis of membrane proteins.     

A substrate of the cell-attachment sequence of fibronectin (Arg-Gly-Asp-Ser) is sufficient to promote transition of arterial smooth muscle cells from a contractile to a synthetic phenotype

Extracellular matrix components strongly influence the differentiated properties of isolated rat arterial smooth muscle cells during in vitro cultivation. The attachment and spreading of the cells on a substrate of fibronectin or a 105-kDa cell-binding fragment of fibronectin are accompanied by a structural and functional transformation, referred to as a transition or modulation from a contractile to a synthetic phenotype. Here, the ability of the cell-attachment sequence of fibronectin, Arg-Gly-Asp-Ser (RGDS), to promote this process was studied. The results demonstrate that freshly isolated smooth muscle cells attached to a substrate of the synthetic peptide Gly-Arg-Gly-Asp-Ser-Cys (GRGDSC) in a specific manner and as well as to substrates of fibronectin and the 105-kDa fragment. Subsequent spreading of the cells on the peptide substrate followed the same kinetics and was as extensive as on fibronectin, even if protein synthesis was blocked by treatment of the cultures with cycloheximide. Like fibronectin, the peptide substrate induced formation of actin filament bundles, again without ongoing protein synthesis. Moreover, it was as efficient as fibronectin in supporting the transition of the cells from a contractile to a synthetic phenotype as analyzed by electron microscopy. Antibodies against the beta subunit of the fibronectin receptor interfered with the attachment, spreading, and fine structural reorganization of the cells in a similar manner on substrates of fibronectin, the 105-kDa fragment, and GRGDSC. Taken together, the findings indicate that the cell-attachment sequence (RGDS) mimics intact fibronectin in promoting a change in the differentiated properties of arterial smooth muscle cells and does so by interacting with a cell surface receptor for fibronectin.     

Calyculin A retraction of mature megakaryocytes proplatelets from embryonic stem cells

Platelets are produced by megakaryocytes (MKs) through proplatelet formation (PPF), or cytoplasmic extensions, in vitro. Through the use of video-enhanced light microscopy, as well as localization of cytoskeletal proteins by confocal microscopy, the reaction of fully mature MK proplatelets, derived from murine embryonic stem cells, to various agents was studied. Calyculin A (protein phosphatase 1/2A inhibitor) treatment induced proplatelet retraction. In MKs with PPF, the expression of actin, myosin IIA, monophosphorylated myosin light chain (MLC-P1), and diphosphorylated myosin light chain (MLC-P2) was diffusely located. Following calyculin A treatment, actin was diffusely localized in retracted MKs and was expressed particularly in the periphery. MLC-P1 was also localized primarily in the periphery; however, MLC-P2 was expressed mostly in the inner area of proplatelets. Protein phosphatase inhibitors may result in increased hyperphosphorylation of localized MLC, which could alter the balance of actomyosin force in a cell, and therefore induce proplatelets retraction.     

Caveolin-1 is required for contractile phenotype expression by airway smooth muscle cells

Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, but the functional role of caveolae and caveolin-1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin-1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)-β(1). As assessed by Western analysis and laser scanning cytometry, caveolin-1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF-β(1) induced profound increases in the contractile phenotype markers sm-α-actin and calponin in cells that also accumulated abundant caveolin-1; however, siRNA or shRNAi inhibition of caveolin-1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF-β(1). The failure by TGF-β(1) to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor-4E binding protein(4E-BP)1 phosphorylation with caveolin-1 knockdown, indicating that caveolin-1 expression promotes TGF-β(1) signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin-1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin-1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin-1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.     

Ex vivo generation of mature and functional human smooth muscle cells differentiated from skeletal myoblasts

We described the ex vivo production of mature and functional human smooth muscle cells (SMCs) derived from skeletal myoblasts. Initially, myoblasts expressed all myogenic cell-related markers such as Myf5, MyoD and Myogenin and differentiate into myotubes. After culture in a medium containing vascular endothelial growth factor (VEGF), these cells were shown to have adopted a differentiated SMC identity as demonstrated by alphaSMA, SM22alpha, calponin and smooth muscle-myosin heavy chain expression. Moreover, the cells cultured in the presence of VEGF did not express MyoD anymore and were unable to fuse in multinucleated myotubes. We demonstrated that myoblasts-derived SMCs (MDSMCs) interacted with endothelial cells to form, in vitro, a capillary-like network in three-dimensional collagen culture and, in vivo, a functional vascular structure in a Matrigel implant in nonobese diabetic-severe combined immunodeficient mice. Based on the easily available tissue source and their differentiation into functional SMCs, these data argue that skeletal myoblasts might represent an important tool for SMCs-based cell therapy.     

Construction of vascular tissues with macro-porous nano-fibrous scaffolds and smooth muscle cells enriched from differentiated embryonic stem cells

Vascular smooth muscle cells (SMCs) have been broadly used for constructing tissue-engineered blood vessels. However, the availability of mature SMCs from donors or patients is very limited. Derivation of SMCs by differentiating embryonic stem cells (ESCs) has been reported, but not widely utilized in vascular tissue engineering due to low induction efficiency and, hence, low SMC purity. To address these problems, SMCs were enriched from retinoic acid induced mouse ESCs with LacZ genetic labeling under the control of SM22α promoter as the positive sorting marker in the present study. The sorted SMCs were characterized and then cultured on three-dimensional macro-porous nano-fibrous scaffolds in vitro or implanted subcutaneously into nude mice after being seeded on the scaffolds. Our data showed that the LacZ staining, which reflected the corresponding SMC marker SM22α expression level, was efficient as a positive selection marker to dramatically enrich SMCs and eliminate other cell types. After the sorted cells were seeded into the three-dimensional nano-fibrous scaffolds, continuous retinoic acid treatment further enhanced the SMC marker gene expression level while inhibited pluripotent maker gene expression level during the in vitro culture. Meanwhile, after being implanted subcutaneously into nude mice, the implanted cells maintained the positive LacZ staining within the constructs and no teratoma formation was observed. In conclusion, our results demonstrated the potential of SMCs derived from ESCs as a promising cell source for therapeutic vascular tissue engineering and disease model applications.     

GM-CSF production from human airway smooth muscle cells is potentiated by human serum

Recent evidence suggests that airway smooth muscle cells (ASMC) actively participate in the airway inflammatory process in asthma. Interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) induce ASMC to release inflammatory mediators in vitro. ASMC mediator release in vivo, however, may be influenced by features of the allergic asthmatic phenotype. We determined whether; (1) allergic asthmatic serum (AAS) modulates ASMC mediator release in response to IL-1beta and TNF-alpha, and (2) IL-1beta/TNF-alpha prime ASMC to release mediators in response to AAS. IL-5 and GM-CSF were quantified by ELISA in culture supernatants of; (1) ASMC pre-incubated with either AAS, nonallergic non-asthmatic serum (NAS) or Monomed (a serum substitute) and subsequently stimulated with IL-1beta and TNF-alpha and (2) ASMC stimulated with IL-1beta/TNF-alpha and subsequently exposed to either AAS, NAS or Monomed. IL-1beta and TNF-alpha induced GM-CSF release in ASMC pre-incubated with AAS was not greater than that in ASMC pre-incubated with NAS or Monomed. IL-1beta and TNF-alpha, however, primed ASMC to release GM-CSF in response to human serum. GM-CSF production following IL-1beta/TNF-alpha and serum exposure (AAS or NAS) was significantly greater than that following IL-1beta/TNF-alpha and Monomed exposure or IL-1beta/TNF-alpha exposure only. Whilst the potentiating effects of human serum were not specific to allergic asthma, these findings suggest that the secretory capacity of ASMC may be up-regulated during exacerbations of asthma, where there is evidence of vascular leakage.