Graphene enhances the cardiomyogenic differentiation of human embryonic stem cells

Graphene has drawn attention as a substrate for stem cell culture and has been reported to stimulate the differentiation of multipotent adult stem cells. Here, we report that graphene enhances the cardiomyogenic differentiation of human embryonic stem cells (hESCs) at least in part, due to nanoroughness of graphene. Large-area graphene on glass coverslips was prepared via the chemical vapor deposition method. The coating of the graphene with vitronectin (VN) was required to ensure high viability of the hESCs cultured on the graphene. hESCs were cultured on either VN-coated glass (glass group) or VN-coated graphene (graphene group) for 21 days. The cells were also cultured on glass coated with Matrigel (Matrigel group), which is a substrate used in conventional, directed cardiomyogenic differentiation systems. The culture of hESCs on graphene promoted the expression of genes involved in the stepwise differentiation into mesodermal and endodermal lineage cells and subsequently cardiomyogenic differentiation compared with the culture on glass or Matrigel. In addition, the culture on graphene enhanced the gene expression of cardiac-specific extracellular matrices. Culture on graphene may provide a new platform for the development of stem cell therapies for ischemic heart diseases by enhancing the cardiomyogenic differentiation of hESCs.     

Wnt proteins promote neuronal differentiation in neural stem cell culture

Wnt signaling is implicated in the control of cell growth and differentiation during CNS development from studies of mouse and chick models, but its action at the cellular level has been poorly understand. In this study, we examine the in vitro function of Wnt signaling in embryonic neural stem cells, dissociated from neurospheres derived from E11.5 mouse telencephalon. Conditioned media containing active Wnt-3a proteins are added to the neural stem cells and its effect on regeneration of neurospheres and differentiation into neuronal and glial cells was examined. Wnt-3a proteins inhibit regeneration of neurospheres, but promote differentiation into MAP2-positive neuronal cells. Wnt-3a proteins also increase the number of GFAP-positive astrocytes but suppress the number of oligodendroglial lineage cells expressing PDGFR or O4. These results indicate that Wnt-3a signaling can inhibit the maintenance of neural stem cells, but rather promote the differentiation of neural stem cells into several cell lineages.     

Polymeric fibrous matrices for substrate-mediated human embryonic stem cell lineage differentiation

Chitosan-based fibrous matrices are prepared to mimic the ECM architecture and elucidate substrate-mediated hESC differentiation due to topographical scale and anisotropy without exogenic morphogens. Fibrous matrices support fewer pluripotent hESCs than films but enable topography-mediated hESC differentiation. Matrices composed of 400 nm and 1.1 μm diameter fibers support increased expression of neural markers indicative of ectodermal commitment while matrices of 200 nm diameter fibers increase expression of osteogenic and hepatic markers indicative of endodermal and mesodermal commitment. The fibrous-mediated hESC differentiation highlights the significant implication of tailored ECM-like substrates for hESC-based therapies.     

Acellular dental matrices promote functional differentiation of ameloblasts

EDTA treatment of post-natal mouse molars made possible the isolation of cell-free dental matrices composed of basal lamina, predentin, dentin and enamel. Trypsin-isolated dental papillae and enamel organs from embryonic-mouse mandibular molars were combined with isolated matrices and cultured in vitro. In such recombinations, functional odontoblasts were never observed. On the other hand, competent preameloblasts in contact with the epithelial side of occlusal predentin overtly differentiated. Matrices treated with guanidine-EDTA or acetic acid were unable to promote the functional differentiation of ameloblasts. These data are discussed in terms of the epitheliomesenchymal interactions involved in odontogenesis.     

Directed differentiation of human mesenchymal stem cells toward a cardiomyogenic fate commitment through formation of cell aggregates

Cell morphology is known to modulate the multipotential lineage commitment of stem cells. We provide a new strategy to induce the early lineage commitment of human mesenchymal stem cells (hMSCs) toward a cardiomyogenic fate through the formation of cell aggregates. A surface-immobilized polyamidoamine dendrimer with fifth generation of dendron structure was used during the culturing of hMSCs. These hMSCs cultured on the G5 surface formed aggregates through active migration and division. More than 22% of cardiac troponin-T (cTnT)-positive (cTnT⁺) cells in aggregates formed on the dendrimer surface; the population formed on the dendrimer surface was higher than that in conventional culture vessel. When cell aggregate was reseeded onto a fresh G5 surface, single cells migrated out of the aggregates, proliferated, and formed new aggregates. This passage method, accompanied with repetitive aggregate dispersion and formation, was applied to cultures over 40 days. The proportion of cTnT⁺ cells increased to 62% by the end of third passage. Our results suggest that culturing hMSCs on G5 surface results in directed commitment of the hMSCs toward a cardiomyocyte-like fate.     

1-磷酸鞘氨醇促进间充质干细胞向心肌分化

为研究1-磷酸鞘氨醇 (Sphingosine-1-phosphate,S1P) 对脐带间充质干细胞 (Umbilical cord mesenchymal stem cells,UC-MSCs) 和脂肪间充质干细胞 (Adipose derived mesenchymal stem cells,AD-MSCs) 向心肌分化的影响,探索其适宜的作用时间和浓度,将UC-MSCs和AD-MSCs接种到培养板,用添加不同浓度S1P的心肌细胞培养液诱导两种干细胞向心肌分化,诱导时间分为7 d、14 d和28 d。采用免疫荧光染色检测心肌特异性蛋白,α-肌动蛋白 (α-actin)、缝隙连接蛋白 (Connexin-43) 以及肌球蛋白重链 (MYH-6) 的表达,并通过共聚焦显微镜和荧光显微镜进行观察;采用MTT分析细胞的活性;膜片钳检测分化细胞的钙瞬变 (此为心肌细胞的功能性指标)。结果表明,S1P与心肌细胞培养液协同作用,能够促进UC-MSCs和AD-MSCs向心肌细胞的分化。并且,随着S1P浓度的增加,促分化作用增强,但细胞活性降低。S1P在心肌细胞培养液中的适宜作用时间为14 d,适宜作用浓度为0.5 μmol/L。而且联合心肌细胞培养液可以使UC-MSCs和AD-MSCs的心肌分化细胞产生钙瞬变,具有类似心肌细胞的功能性。S1P能够与心肌细胞培养液协同作用,促进UC-MSCs和AD-MSCs的心肌功能性分化。     

Enhanced cardiomyogenic lineage differentiation of adult bone-marrow-derived stem cells grown on cardiogel

The extracellular matrix (ECM) and its components are known to promote growth and cellular differentiation in vitro. Cardiogel, a three-dimensional extracellular matrix derived from cardiac fibroblasts, is evaluated for its cardiomyogenic-differentiation-inducing potential on bone-marrow-derived stem cells (BMSC). BMSC from adult mice were grown on cardiogel and induced to differentiate into specific lineages that were validated by morphological, phenotypic and molecular assays. The data revealed that the cardiogel enhanced cardiomyogenic and adipogenic differentiation and relegated osteogenic differentiation following specific induction. More importantly, increased cardiomyogenic differentiation was also observed following BMSC growth on cardiogel without specific chemical (5-azacytidine) induction. This is the first report of an attempt to use cardiogel as a biomaterial on which to achieve cardiomyogenic differentiation of BMSC without chemical induction. Our study suggests that cardiogel is an efficient extracellular matrix that enhances the cardiomyogenic differentiation of BMSC and that it can therefore be used as a scaffold for cardiac tissue regeneration.     

N-cadherin is essential for retinoic acid-mediated cardiomyogenic differentiation in mouse embryonic stem cells

Contraction forces developed by cardiomyocytes are transmitted across the plasma membrane through end-to-end connections between the myocytes, called intercalated disks, which enable the coordinated contraction of heart muscle. A component of the intercalated disk, the adherens junction, consists of the cell adhesion molecule, N-cadherin. Embryos lacking N-cadherin die at mid-gestation from cardiovascular abnormalities. We have evaluated the role of N-cadherin in cardiomyogenesis using N-cadherin-null mouse embryonic stem (ES) cells grown as embryoid bodies (EBs) in vitro. Myofibrillogenesis, the spatial orientation of myofibers, and intercellular contacts including desmosomes were normal in N-cadherin-null ES cell-derived cardiomyocytes. The effect of retinoic acid (RA), a stage and dose-dependent cardiogenic factor, was assessed in differentiating ES cells. all-trans (at) RA increased the number of ES cell-derived cardiomyocytes by approximately 3-fold (at 3 x 10(-9) M) in wt EBs. However, this effect was lost in N-cadherin-null EBs. In the presence of supplemented at-RA, the emergence of spontaneously beating cardiomyocytes appeared to be delayed and slightly less efficient in N-cadherin-null compared with wt and heterozygous EBs (frequencies of EBs with beating activity at 5 days: 54+/-18% vs. 96+/-0.5%, and 93+/-7%, respectively; peak frequencies of EBs with beating activity: 83+/-8% vs. 96+/-0.5% and 100%, respectively). In conclusion, cardiomyoyctes differentiating from N-cadherin-null ES cells in vitro show normal myofibrillogenesis and intercellular contacts, but impaired responses to early cardiogenic effects mediated by at-RA. These results suggest that N-cadherin may be essential for RA-induced cardiomyogenesis in mouse ES cells in vitro.     

Mesenchymal stem cell responses to bone-mimetic electrospun matrices composed of polycaprolactone, collagen I and nanoparticulate hydroxyapatite

The performance of biomaterials designed for bone repair depends, in part, on the ability of the material to support the adhesion and survival of mesenchymal stem cells (MSCs). In this study, a nanofibrous bone-mimicking scaffold was electrospun from a mixture of polycaprolactone (PCL), collagen I, and hydroxyapatite (HA) nanoparticles with a dry weight ratio of 50/30/20 respectively (PCL/col/HA). The cytocompatibility of this tri-component scaffold was compared with three other scaffold formulations: 100% PCL (PCL), 100% collagen I (col), and a bi-component scaffold containing 80% PCL/20% HA (PCL/HA). Scanning electron microscopy, fluorescent live cell imaging, and MTS assays showed that MSCs adhered to the PCL, PCL/HA and PCL/col/HA scaffolds, however more rapid cell spreading and significantly greater cell proliferation was observed for MSCs on the tri-component bone-mimetic scaffolds. In contrast, the col scaffolds did not support cell spreading or survival, possibly due to the low tensile modulus of this material. PCL/col/HA scaffolds adsorbed a substantially greater quantity of the adhesive proteins, fibronectin and vitronectin, than PCL or PCL/HA following in vitro exposure to serum, or placement into rat tibiae, which may have contributed to the favorable cell responses to the tri-component substrates. In addition, cells seeded onto PCL/col/HA scaffolds showed markedly increased levels of phosphorylated FAK, a marker of integrin activation and a signaling molecule known to be important for directing cell survival and osteoblastic differentiation. Collectively these results suggest that electrospun bone-mimetic matrices serve as promising degradable substrates for bone regenerative applications.     

Auditory hair cell explant co-cultures promote the differentiation of stem cells into bipolar neurons

Auditory neurons, the target neurons of the cochlear implant, degenerate following a sensorineural hearing loss. The goal of this research is to direct the differentiation of embryonic stem cells (SCs) into bipolar auditory neurons that can be used to replace degenerating neurons in the deafened mammalian cochlea. Successful replacement of auditory neurons is likely to result in improved clinical outcomes for cochlear implant recipients. We examined two post-natal auditory co-culture models with and without neurotrophic support, for their potential to direct the differentiation of mouse embryonic SCs into characteristic, bipolar, auditory neurons. The differentiation of SCs into neuron-like cells was facilitated by co-culture with auditory neurons or hair cell explants, isolated from post-natal day five rats. The most successful combination was the co-culture of hair cell explants with whole embryoid bodies, which resulted in significantly greater numbers of neurofilament-positive, neuron-like cells. While further characterization of these differentiated cells will be essential before transplantation studies commence, these data illustrate the effectiveness of post-natal hair cell explant co-culture, at providing valuable molecular cues for directed differentiation of SCs towards an auditory neuron lineage.     

Patterning stem cell differentiation

Regulation of cell differentiation and assembly remains a fundamental question in developmental biology. Now, a report from the Chen laboratory (Ruiz and Chen, 2008) describes an approach that represents a major step toward a more profound understanding of the geometric-force control of stem cell differentiation.     

Synthetic sulfonyl-hydrazone-1 positively regulates cardiomyogenic microRNA expression and cardiomyocyte differentiation of induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) are obtained from adult cells through overexpression of pluripotency factors. iPSCs share many features with embryonic stem cells (ESCs), circumventing ethical issues, and, noteworthy, match donor's genotype. iPSCs represent therefore a valuable tool for regenerative medicine. Cardiac differentiation of ESCs can be enhanced via microRNAs (miRNAs) and small chemical compounds, which probably act as chromatin remodelers. Cardiomyogenic potential of iPSCs is currently intensely investigated for cell therapy or in vitro drug screening and disease modeling. However, influences of small compounds on iPSC‐related cardiomyogenesis have not yet been investigated in details. Here, we compared the effects of two small molecules, bis‐peroxo‐vanadium (bpV) and sulfonyl‐hydrazone‐1 (SHZ) at varying concentrations, during cardiac differentiation of murine iPSCs. SHZ (5 µM) enhanced specific marker expression and cardiomyocyte yield, without loss of cell viability. In contrast, bpV showed negligible effects on cardiac differentiation rate and appeared to induce Casp3‐dependent apoptosis in differentiating iPSCs. Furthermore, SHZ‐treated iPSCs were able to increase beating foci rate and upregulate early and late cardiomyogenic miRNA expression (miR‐1, miR‐133a, and miR‐208a). Thus, our results demonstrate that small chemical compounds, such as SHZ, can constitute a novel and clinically feasible strategy to improve iPSC‐derived cardiac differentiation. J. Cell. Biochem. 112: 2006–2014, 2011. © 2011 Wiley‐Liss, Inc.     

Cell-cell interactions promote mammary epithelial cell differentiation

Mammary epithelium differentiates in a stromal milieu of adipocytes and fibroblasts. To investigate cell-cell interactions that may influence mammary epithelial cell differentiation, we developed a co-culture system of murine mammary epithelium and adipocytes and other fibroblasts. Insofar as caseins are specific molecular markers of mammary epithelial differentiation, rat anti-mouse casein monoclonal antibodies were raised against the three major mouse casein components to study this interaction. Mammary epithelium from mid-pregnant mice was plated on confluent irradiated monolayers of 3T3-L1 cells, a subclone of the Swiss 3T3 cell line that differentiates into adipocytes in monolayer culture and other cell monolayers (3T3-C2 cells, Swiss 3T3 cells, and human foreskin fibroblasts). Casein was synthesized by mammary epithelium only in the presence of co-cultured cells and the lactogenic hormone combination of insulin, hydrocortisone, and prolactin. Synthesis and accumulation of alpha-, beta-, and gamma-mouse casein within the epithelium was shown by immunohistochemical staining of cultured cells with anti-casein monoclonal antibodies, and the specificity of the immunohistochemical reaction was demonstrated using immunoblots. A competitive immunoassay was used to measure the amount of casein secreted into the culture medium. In a 24-h period, mammary epithelium co-cultured with 3T3-L1 cells secreted 12-20 micrograms beta-casein per culture dish. There was evidence of specificity in the cell-cell interaction that mediates hormone-dependent casein synthesis. Swiss 3T3 cells, newborn foreskin fibroblasts, substrate-attached material ("extracellular matrix"), and tissue culture plastic did not support casein synthesis, whereas monolayers of 3T3-L1 and 3T3-C2 cells, a subclone of Swiss 3T3 cells that does not undergo adipocyte differentiation, did. We conclude that interaction between mammary epithelium and other specific nonepithelial cells markedly influences the acquisition of hormone sensitivity of the epithelium and hormone-dependent differentiation.