抑制NHE1活性对干细胞向心肌分化的影响

Na /H 交换蛋白1(NHE1)在心肌细胞发育过程中发挥重要的调节功能.为深入探索NHEl活性对干细胞向心肌分化过程中产生的影响,采用二甲基亚砜(DMSO)诱导P19干细胞向心肌细胞分化,同时在培养液中添加NHE1抑制荆EMD87580,对诱导后形成的类胚体进行检测.通过细胞形态观察、免疫组织化学染色及检测心肌特异表达基因等方法证明,经诱导形成的类胚体贴壁生长后,会向心肌细胞分化并出现跳动细胞团.而经过抑制剂处理的P19干细胞尽管能够形成类胚体且贴壁培养后细胞仍具有增殖活力,细胞团周边也较整齐,但未出现向心肌细胞分化的现象.这一结果表明,抑制NHE1的活性,能够影响P19干细胞向心肌细胞的分化作用.     

Synthesis of Biomimetic Superhydrophobic Surface through Electrochemical Deposition on Porous Alumina

The superhydrophobicity of plant leaves is a benefit of the hierarchical structures of their surfaces. These structures have been imitated in the creation of synthetic surfaces. In this paper, a novel process for fabrication of biomimetic hierarchical structures by electrochemical deposition of a metal on porous alumina is described. An aluminum specimen was anodically oxidized to obtain a porous alumina template, which was used as an electrode to fabricate a surface with micro structures through electrochemical deposition of a metal such as nickel and copper after the enlargement of pores. Astonishingly, a hierarchical structure with nanometer pillars and micrometer clusters was synthesized in the pores of the template. The nanometer pillars were determined by the nanometer pores. The formation of micrometer clusters was related to the thin walls of the pores and the crystallization of the metal on a flat surface. From the as-prepared biomimetic surfaces, lotus-leaf-like superhydrophobic surfaces with nickel and copper deposition were achieved.     

Beta-adrenergic and muscarinic modulation of human embryonic stem cell-derived cardiomyocytes.

BACKGROUND: Embryonic stem cells provide the most promising tool for cell replacement therapy including transplantation of human embryonic stem (hES) cell- derived cardiomyocytes in the infarcted area of the heart. Here we provide data for differentiation of cardiomyocytes from hES cells and firstly describe their hormonal modulation. METHODS: Using Micro-Electrode Arrays as a novel electrical mapping technique of beating cardiomyocyte clusters within whole hES cell aggregates, we were able to measure the field potential generation and morphology changes during hormonal modulation. RESULTS: We found that isoproterenol provokes, similar to the mouse ES cell system, a strong positive chronotropic effect with an EC50 of around 10(-8) M. Moreover, isoproterenol stimulated with a higher EC50 value the slow field potential amplitude, FP(slow), indicating a stimulation of Ca2+ channels in ventricular-like ES cell-derived cardiomyocytes which is shown to be clearly independent from frequency modulation. In contrast, carbachol (10 microM) produced a transient negative chronotropic effect but had no effect on FP(slow). CONCLUSION: The Micro-Electrode system allows measurement of ionic channel modulation and chronotropic responsiveness in a pharmacological screening setup. Moreover, all our data indicate that cardiomyocytes derived from human embryonic stem cells exhibit a physiological response to the major hormones of the vegetative nervous system and might therefore serve as an ideal candidate for the use in cell replacement strategies.     

Cardiac potential of stem cells from whole human umbilical cord tissue

We investigated the role of stem cells from human umbilical cord tissue in cardiomyocyte regeneration. The umbilical cord stem cells were initially characterized and differentiated in a myocardial differentiation medium containing 5‐azacytidine for 24 h. Differentiation into cardiomyocytes was determined by expression of cardiac specific markers, like cardiac α‐actin, connexin43, myosin, Troponin T, and ultrastructural analysis. In vivo, the transplanted umbilical cord stem cells were sprouting from local injection and differentiated into cardiomyocyte‐like cells in a rat myocardial infarction model. Echocardiography revealed increasing left ventricular function after umbilical cord stem cell transplantation. These results demonstrate that umbilical cord stem cells can differentiate into cardiomyocyte‐like cells both in vitro and in vivo. Therefore, human umbilical cord might represent a source of stem cells useful for cellular therapy and myocardial tissue engineering. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon. J. Cell. Biochem. 107: 926–932, 2009. © 2009 Wiley‐Liss, Inc.     

A non-destructive culturing and cell sorting method for cardiomyocytes and neurons using a double alginate layer

A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES) cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture.     

AFM and impedance spectroscopy characterization of the immobilization of antibodies on indium-tin oxide electrode through self-assembled monolayer of epoxysilane and their capture of Escherichia coli O157:H7

The microscopic surface molecular structures and macroscopic electrochemical impedance properties of the epoxysilane monolayer and anti-Escherichia coli antibody layer on an indium-tin oxide (ITO) electrode surface were studied in this paper. Characterization of stepwise changes in microscopic features of the surfaces and electrochemical properties upon the formation of each layer were carried out using both atomic force microscopy (AFM) and electrochemical impedance spectroscopy in the presence of [Fe(CN)6](3-/4-) as a redox couple. AFM images of the self-assembled monolayer (SAM) evidenced the dense, complete, and homogeneous morphology of the epoxysilane monolayer on the ITO surface. The uniformity of the epoxysilane SAM allowed antibodies to attach to the epoxy surface groups of the silanes in a similarly uniform fashion. The effects of epoxysilane monolayer and the antibody layer on the electrochemical properties of the electrode were quantitatively analyzed in terms of double layer capacitance, electron transfer resistance, Warburg impedance and solution resistance using Randles model as the equivalent circuit. It was demonstrated that the epoxysilane monolayer and the antibody layer act as barriers for the electron transfer between the electrode surface and the redox species in the solution, resulting in most significant increases in the electron transfer resistance compared to all the electric elements. Immunoreaction with E. coli O157:H7 cells demonstrated specific recognition of the immobilized anti-E. coli antibodies as evidenced by AFM imaging and impedance spectroscopy. It was found that the binding of E. coli cells mainly affected the electron transfer resistance and Warburg impedance.     

Cardiomyocyte marker expression in a human lymphocyte cell line using mouse cardiomyocyte extract

Cell transplantation shows potential for the treatment of cardiac diseases. Embryonic stem cells, cord blood and mesenchymal stem cells have been suggested as sources for transplantation therapy. Because of some technical limitations with the use of stem cells, transdifferentiation of fully differentiated cells is a potentially useful alternative. We investigated whether human peripheral blood cells could transdifferentiate into cardiomyocyte. Transdifferentiation was induced in a human B lymphocyte cell line (Raji). Cardiomyocyte extract was prepared from adult mouse cardiomyocytes. The cells were treated with 5-aza-2-deoxycytidine and trichostatin A, permeabilized with streptolysin O, and exposed to the mouse cardiomyocyte extract. They were cultured for 10 days, 3 weeks and 4 weeks. Cardiomyocyte markers were detected with immunohistochemistry and flow cytometry. Immunocytochemistry revealed that some cells expressed myosin heavy chain, α-actinin and cardiac troponin T after 3 and 4 weeks. Flow cytometry confirmed these data. In cells exposed to trichostatin A and 5-aza-2-deoxycytidine and permeabilized in the presence of the cardiomyocyte extract, troponin T expression was seen in 3.53% of the cells and 3.11% of them expressed α-actinin. After exposure to the cardiomyocyte extract, some permeabilized cells adhered to the plate loosely; however, the morphology did not change significantly, and they continued to show a rounded shape after 4 weeks. Our treated lymphocytes expressed cardiomyocyte markers. Our results suggest that lymphocytes may be useful in future research as a source of cells for reprogramming procedures.     

Cardiomyocyte precursors and telocytes in epicardial stem cell niche: electron microscope images

A highly heterogeneous population of stem and progenitor cells has been described by light immunohistochemistry in the mammalian adult heart, but the ultrastructural identity of cardiac stem cells remains unknown. Using electron microscopy, we demonstrate the presence of cells with stem features in the adult mouse heart. These putative cardiac stem cells are small (6–10 μm), round cells, with an irregular shaped nucleus, large nucleolus, few endoplasmic reticulum cisternae and mitochondria, but numerous ribosomes. Stem cells located in the epicardial stem cell niche undergo mitosis and apoptosis. Cells with intermediate features between stem cells and cardiomyocyte progenitors have also been seen. Moreover, electron microscopy showed that cardiomyocyte progenitors were added to the peripheral working cardiomyocytes. Telocytes make a supportive interstitial network for stem cells and progenitors in the stem cell niche. This study enhances the hypothesis of a unique type of cardiac stem cell and progenitors in different stages of differentiation. In our opinion, stem cells, cardiomyocyte progenitors and telocytes sustain a continuous cardiac renewal process in the adult mammalian heart.     

The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis

In order to examine the spatial organisation of stem cells and their progeny in human epidermis, we developed a method for whole-mount epidermal immunofluorescence labelling using high surface beta1 integrin expression as a stem cell marker. We confirmed that there are clusters of high beta1 integrin-expressing cells at the tips of the dermal papillae in epidermis from several body sites, whereas alpha6 integrin expression is more uniform. The majority of actively cycling cells detected by Ki67 or bromodeoxyuridine labelling were found in the beta1 integrin-dull, transit amplifying population and integrin-negative, keratin 10-positive cells left the basal layer exclusively from this compartment. When we examined p53-positive clones in sun-exposed epidermis, we found two types of clone that differed in size and position in a way that was consistent with the founder cell being a stem or transit amplifying cell. The patterning of the basal layer implies that transit amplifying cells migrate over the basement membrane away from the stem cell clusters. In support of this, isolated beta1 integrin-dull keratinocytes were more motile on type IV collagen than beta1 integrin-bright keratinocytes and EGFP-labelled stem cell clones in confluent cultured sheets were compact, whereas transit amplifying clones were dispersed. The combination of whole-mount labelling and lineage marking thus reveals features of epidermal organisation that were previously unrecognised.     

Stimulation of human epidermal differentiation by delta-notch signalling at the boundaries of stem-cell clusters

BACKGROUND: Human epidermis is renewed throughout life from stem cells in the basal layer of the epidermis. Signals from the surrounding keratinocytes influence the differentiation of the stem cells, but the nature of the signals is unknown. In many developing tissues, signalling mediated by the transmembrane protein Delta1 and its receptor Notch1 inhibits differentiation. Here, we investigated the role of Delta-Notch signalling in postnatal human epidermis. RESULTS: Notch1 expression was found in all living epidermal layers, but Delta1 expression was confined to the basal layer of the epidermis, with highest expression in those regions where stem cells reside. By overexpressing Delta1 or Delta(T), a truncated form of Delta1, in primary human keratinocytes and reconstituting epidermal sheets containing mixtures of Delta-overexpressing cells and wild-type cells, we found that cells expressing high levels of Delta1 or Delta(T) failed to respond to Delta signals from their neighbours. In contrast, wild-type keratinocytes that were in contact with neighbouring cells expressing Delta1 were stimulated to leave the stem-cell compartment and initiate terminal differentiation after a few rounds of division. Delta1 promoted keratinocyte cohesiveness, whereas Delta(T) did not. CONCLUSIONS: We propose that high Delta1 expression by epidermal stem cells has three effects: a protective effect on stem cells by blocking Notch signalling; enhanced cohesiveness of stem-cell clusters, which may discourage intermingling with neighbouring cells; and signalling to cells at the edges of the clusters to differentiate. Notch signalling in epidermal stem cells thus differs from other progenitor cell populations in promoting, rather than suppressing, differentiation.     

Chemical and physical stimuli induce cardiomyocyte differentiation from stem cells

In this study we investigated cardiomyocyte differentiation of rat bone marrow-mesenchymal stem cells (BM-MSCs) by treating the stem cells with conditions mimicking that of myocardial infarction. The extract from infarcted rat myocardium contained the biochemical factors arising after infarction. The cardiac contraction and relaxation were simulated by applying 4% strain at 1 Hz to the stem cells. We found that the extract from infarcted myocardium or 4% strain each alone could induce cardiomyocyte differentiation of BM-MSCs, as shown by expression of cardiomyocyte-specific genes including α-actin, connexin 43, Nkx2.5, MEF2c, GATA4, α-MHC, and Troponin I. Furthermore, a combination of the extract and 4% strain had stronger effects on cardiomyocyte differentiation than what either treatment alone had. Our results suggest that this in vitro model system simulates the local cardiac environment cues after infarction and may be useful in identifying the biochemical and physical factors involved in cardiomyocyte differentiation.     

In vitro inhibitory effect of interferon on colony formation of myeloma stem cells

Summary The inhibitory effect of interferon on colony formation of myeloma stem cells in two layer plasma clot-soft agar cultures was studied. Human lymphoblast interferon inhibited in therapeutically attainable concentrations myeloma stem cell proliferation in 50% and human fibroblast interferon in 23% of the 14 myeloma patients in whom in vitro colony formation could be achieved. In interferon-sensitive patients the numbers of myeloma stem cell clusters and colonies were decreased to 34.4%–54.9% of control cultures. In addition, maturation of myeloma stem cells in differentiated plasma cells was reduced by interferon in most of these cases.     

Stem surface injuries of 20 species of succulent Euphorbia (Euphorbiacae) from South Africa

Cactus and succulent Euphorbia plants have green stems that are exposed to sunlight over long periods. Previous research demonstrated that barking injuries occur to stem surfaces of 20 species of cacti in the Americas. For these 20 cactus species the amounts of injuries were related to annual amounts of sunlight exposure on surfaces. Cacti with many injuries had high mortality rates. Data herein show that stem surfaces of 20 species of Euphorbia in South Africa have identical surface injuries as for cacti. Euphorbia plants at 30°S show a mean ratio of scale and bark injuries of 3.5:1 of equatorial-facing to polar-facing surfaces, a ratio is similar to the 4:1 of annual sunlight exposure ratio at that latitude. Results show that Euphorbia stems have injuries on younger tissues than cactus species. Thus, Euphorbia stems are more sensitive than cactus stems. Euphorbia species with most stem injuries had smooth (non-undulate) surfaces, a one-celled epidermal cell layer only, cortex cells that abutted the epidermis, and initial injuries that involved only epidermal cells. This is the first report of identical stem surface injuries to Euphorbia species that are identical to surface injuries to cactus species worldwide. Moreover, the amounts on stem injuries on Euphorbia and long-lived, columnar cactus species are highly correlated with relative amounts of sunlight on stem surfaces over the annual cycle. These results suggest that these species are good bio-indicators of annual sunlight exposure.     

Generation and Characterization of Functional Cardiomyocytes Derived from Human T Cell-Derived Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have been proposed as novel cell sources for genetic disease models and revolutionary clinical therapies. Accordingly, human iPSC-derived cardiomyocytes are potential cell sources for cardiomyocyte transplantation therapy. We previously developed a novel generation method for human peripheral T cell-derived iPSCs (TiPSCs) that uses a minimally invasive approach to obtain patient cells. However, it remained unknown whether TiPSCs with genomic rearrangements in the T cell receptor (TCR) gene could differentiate into functional cardiomyocyte in vitro. To address this issue, we investigated the morphology, gene expression pattern, and electrophysiological properties of TiPSC-derived cardiomyocytes differentiated by floating culture. RT-PCR analysis and immunohistochemistry showed that the TiPSC-derived cardiomyocytes properly express cardiomyocyte markers and ion channels, and show the typical cardiomyocyte morphology. Multiple electrode arrays with application of ion channel inhibitors also revealed normal electrophysiological responses in the TiPSC-derived cardiomyocytes in terms of beating rate and the field potential waveform. In this report, we showed that TiPSCs successfully differentiated into cardiomyocytes with morphology, gene expression patterns, and electrophysiological features typical of native cardiomyocytes. TiPSCs-derived cardiomyocytes obtained from patients by a minimally invasive technique could therefore become disease models for understanding the mechanisms of cardiac disease and cell sources for revolutionary cardiomyocyte therapies.     

Fibroblast growth factor-10 promotes cardiomyocyte differentiation from embryonic and induced pluripotent stem cells

Background

The fibroblast growth factor (FGF) family is essential to normal heart development. Yet, its contribution to cardiomyocyte differentiation from stem cells has not been systemically studied. In this study, we examined the mechanisms and characters of cardiomyocyte differentiation from FGF family protein treated embryonic stem (ES) cells and induced pluripotent stem (iPS) cells.

Methodology/Principal Findings

We used mouse ES cells stably transfected with a cardiac-specific α-myosin heavy chain (αMHC) promoter-driven enhanced green fluorescent protein (EGFP) and mouse iPS cells to investigate cardiomyocyte differentiation. During cardiomyocyte differentiation from mouse ES cells, FGF-3, -8, -10, -11, -13 and -15 showed an expression pattern similar to the mesodermal marker Brachyury and the cardiovascular progenitor marker Flk-1. Among them, FGF-10 induced cardiomyocyte differentiation in a time- and concentration-dependent manner. FGF-10 neutralizing antibody, small molecule FGF receptor antagonist PD173074 and FGF-10 and FGF receptor-2 short hairpin RNAs inhibited cardiomyocyte differentiation. FGF-10 also increased mouse iPS cell differentiation into cardiomyocyte lineage, and this effect was abolished by FGF-10 neutralizing antibody or PD173074. Following Gene Ontology analysis, microarray data indicated that genes involved in cardiac development were upregulated after FGF-10 treatment. In vivo, intramyocardial co-administration of FGF-10 and ES cells demonstrated that FGF-10 also promoted cardiomyocyte differentiation.

Conclusion/Significance

FGF-10 induced cardiomyocyte differentiation from ES cells and iPS cells, which may have potential for translation into clinical applications.     

Atomic force mechanobiology of pluripotent stem cell-derived cardiomyocytes

We describe a method using atomic force microscopy (AFM) to quantify the mechanobiological properties of pluripotent, stem cell-derived cardiomyocytes, including contraction force, rate, duration, and cellular elasticity. We measured beats from cardiomyocytes derived from induced pluripotent stem cells of healthy subjects and those with dilated cardiomyopathy, and from embryonic stem cell lines. We found that our AFM method could quantitate beat forces of single cells and clusters of cardiomyocytes. We demonstrate the dose-responsive, inotropic effect of norepinephrine and beta-adrenergic blockade of metoprolol. Cardiomyocytes derived from subjects with dilated cardiomyopathy showed decreased force and decreased cellular elasticity compared to controls. This AFM-based method can serve as a screening tool for the development of cardiac-active pharmacological agents, or as a platform for studying cardiomyocyte biology.     

Making cardiomyocytes: How mechanical stimulation can influence differentiation of pluripotent stem cells

Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have promise in regenerative medicine for a variety of applications. Their potential in the treatment of cardiovascular disease is of particular interest due to its severity and prevalence. In order to be successful for cell therapy, PSCs must be pre‐differentiated into cardiomyocytes to prevent teratoma formation in vivo. Current methods focus on the supplementation of soluble factors to culture medium to drive differentiation into mesodermal lineages; however, these methods are costly with varying cardiomyocyte yields. Since cardiomyocytes are exposed to dynamic environments in vivo, there is potential in using mechanical stimulation to further drive differentiation in vitro. In this review, we will describe the most recent developments in how mechanical stimulation, including fluid shear, cyclic strain, and magnetically mediated strain, can guide cardiomyogenesis in PSCs. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1089–1096, 2013     

Hypoxia influences generation and propagation of electrical activity in embryonic cardiomyocyte clusters

The influence of tissue hypoxia on the generation and propagation of excitation was studied in spontaneously beating embryonic cardiomyocyte clusters grown in eight 9-12 days old embryoid bodies. Within the embryoid bodies one to three separately active clusters of cardiomyocytes were found, each having its own pacemaker cell. Lowering of tissue PO(2) caused bradycardia as well as arrhythmia in all embryoid bodies investigated. The mean frequency of the extracellularly recorded action potentials decreased under conditions of pronounced hypoxia from a mean of 1.4-1.8 Hz to below 0.8 Hz. In three embryoid bodies hypoxia-sensitive as well as hypoxia-tolerant cardiomyocyte clusters were found. The hypoxia-insensitive cardiomyocytes showed a low frequency of spontaneous activity. In addition to the observed changes in the generation of excitation, tissue hypoxia caused an approximately 60% reduction in the velocity of conduction within the cardiomyocyte clusters. Moreover, in at least one of the eight experiments propagation failure with an incomplete block in spread of excitation was observed. All hypoxia-induced effects on generation and propagation of embryonic cardiomyocyte excitation were completely reversible after reoxygenation.     

Epicuticular wax structures on stems and comparison between stems and leaves – A survey

The cuticle, forming the outermost layer of plant tissues and being in direct contact with the environment, consists of waxes and cutin. Waxes are hydrophobic substances that are divided in two groups: intra- and epicuticular, depending on their localisation. Epicuticular waxes appear as smooth coverings, however, many plants also produce superimposed wax structures of a crystalline nature. While studies of waxes have almost exclusively focused on leaves, here a survey of epicuticular wax structures on stems is presented. The stem surface of 343 higher plant taxa, representing 80 families, was examined using scanning electron microscopy. The adaxial and abaxial surfaces of leaves of 319 taxa were also examined to determine the relationship between wax structures on stems and leaves. Wax structures are classified, described and discussed. The results of the study indicate that stems exhibit the same main wax crystal types that have been described for leaves. Seventy percent of the examined taxa produced wax crystals on their stems. In ∼24% of the taxa, wax crystals were absent on leaves and found only on stems. In plant taxa that produce wax crystals, 40% exhibit the same type on either side of their leaves and on their stem. However, a much stronger morphological similarity exists between crystal shapes present on the adaxial and abaxial surfaces of leaves than between those present on the stem and those on leaves. In general, these observations suggest that stems are quite different than leaves in terms of their epicuticular wax structures.     

Endothelial cells regulate cardiomyocyte development from embryonic stem cells

The molecules and environment that direct pluripotent stem cell differentiation into cardiomyocytes are largely unknown. Here, we determined a critical role of receptor tyrosine kinase, EphB4, in regulating cardiomyocyte generation from embryonic stem (ES) cells through endothelial cells. The number of spontaneous contracting cardiomyocytes, and the expression of cardiac‐specific genes, including α‐MHC and MLC‐2V, was significantly decreased in EphB4‐null ES cells. EphB4 was expressed in endothelial cells underneath contracting cardiomyocytes, but not in cardiomyocytes. Angiogenic inhibitors, including endostatin and angiostatin, inhibited endothelial cell differentiation and diminished cardiomyogenesis in ES cells. Generation of functional cardiomyocytes and the expression of cardiac‐specific genes were significantly enhanced by co‐culture of ES cells with human endothelial cells. Furthermore, the defects of cardiomyocyte differentiation in EphB4‐deficient ES cells were rescued by human endothelial cells. For the first time, our study demonstrated that endothelial cells play an essential role in facilitating cardiomyocyte differentiation from pluripotent stem cells. EphB4 signaling is a critical component of the endothelial niche to regulate regeneration of cardiomyocytes. J. Cell. Biochem. 111: 29–39, 2010. © 2010 Wiley‐Liss, Inc.