Microgrooved Surface Modulates Neuron Differentiation in Human Embryonic Stem Cells.

Stem cell-based therapies have drawn intensive attention in the neuronal regenerative fields. Several studies have revealed that stem cells can serve as an inexhaustible source for neurons for transplantation therapies. However, generation of neurons and directionality has not yet been fully investigated. Herein, we investigate the mechanical ramifications of surface topography on human embryonic cell differentiation. Microgrooved surfaces with various pitches were applied to modulate the neuron differentiation. Our protocol showed that neuron differentiation increased as grove pitch decreased. The results indicated that 2 µm microgrooves can improve neuron growth by ~1.7-fold. Our results indicate the importance of mechanotransduction on neuronal differentiation and highlight the feasibility of manipulating the neuronal differentiation with surface topography, providing new perspectives for accommodating clinical transplantation.

Ocean warming-acidification synergism undermines dissolved organic matter assembly.

Understanding the influence of synergisms on natural processes is a critical step toward determining the full-extent of anthropogenic stressors. As carbon emissions continue unabated, two major stressors--warming and acidification--threaten marine systems on several scales. Here, we report that a moderate temperature increase (from 30°C to 32°C) is sufficient to slow--even hinder--the ability of dissolved organic matter, a major carbon pool, to self-assemble to form marine microgels, which contribute to the particulate organic matter pool. Moreover, acidification lowers the temperature threshold at which we observe our results. These findings carry implications for the marine carbon cycle, as self-assembled marine microgels generate an estimated global seawater budget of ~1016 g C. We used laser scattering spectroscopy to test the influence of temperature and pH on spontaneous marine gel assembly. The results of independent experiments revealed that at a particular point, both pH and temperature block microgel formation (32°C, pH 8.2), and disperse existing gels (35°C). We then tested the hypothesis that temperature and pH have a synergistic influence on marine gel dispersion. We found that the dispersion temperature decreases concurrently with pH: from 32°C at pH 8.2, to 28°C at pH 7.5. If our laboratory observations can be extrapolated to complex marine environments, our results suggest that a warming-acidification synergism can decrease carbon and nutrient fluxes, disturbing marine trophic and trace element cycles, at rates faster than projected.

Accelerated neuronal differentiation toward motor neuron lineage from human embryonic stem cell line (H9).

Motor neurons loss plays a pivotal role in the pathoetiology of various debilitating diseases such as, but not limited to, amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, pseudobulbar palsy, and spinal muscular atrophy. However, advancement in motor neuron replacement therapy has been significantly constrained by the difficulties in large-scale production at a cost-effective manner. Current methods to derive motor neuron heavily rely on biochemical stimulation, chemical biological screening, and complex physical cues. These existing methods are seriously challenged by extensive time requirements and poor yields. An innovative approach that overcomes prior hurdles and enhances the rate of successful motor neuron transplantation in patients is of critical demand. Iron, a trace element, is indispensable for the normal development and function of the central nervous system. Whether ferric ions promote neuronal differentiation and subsequently promote motor neuron lineage has never been considered. Here, we demonstrate that elevated iron concentration can drastically accelerate the differentiation of human embryonic stem cells (hESCs) toward motor neuron lineage potentially via a transferrin mediated pathway. HB9 expression in 500 nM iron-treated hESCs is approximately twofold higher than the control. Moreover, iron treatment generated more matured and functional motor neuron-like cells that are ∼1.5 times more sensitive to depolarization when compared to the control. Our methodology renders an expedited approach to harvest motor neuron-like cells for disease, traumatic injury regeneration, and drug screening.

Determine the quality of human embryonic stem colonies with laser light scattering patterns.

BACKGROUND: With the prompt developments of regenerative medicine, the potential clinical applications of human embryonic stem cells have attracted intense attention. However, the labor-intensive and complex manual cell selection processes required during embryonic stem cell culturing have seriously limited large-scale production and broad applications. Thus, availability of a label-free, non-invasive platform to replace the current cumbersome manual selection has become a critical need. RESULTS: A non-invasive, label-free, and time-efficient optical platform for determining the quality of human embryonic stem cell colonies was developed by analyzing the scattering signals from those stem cell colonies. Additionally, confocal microscopy revealed that the cell colony morphology and surface structures were correlated with the resulting characteristic light scattering patterns. Standard immunostaining assay (Oct-4) was also utilized to validate the quality-determination from this light scattering protocol. The platform developed here can therefore provide identification accuracy of up to 87% for colony determination. CONCLUSIONS: Our study here demonstrated that light scattering patterns can serve as a feasible alternative approach to replace conventional manual selection for human embryonic stem cell cultures.

Activated charcoal composite biomaterial promotes human embryonic stem cell differentiation toward neuronal lineage.

Transplantation of biomaterial scaffolds encasing human embryonic stem cells (hESCs) has been proposed as a clinical therapy for various neurological lesions and disorders. In light of recent developments, artificially synthesized carbon-based biomaterials such as carbon nanotubes and graphene have demonstrated feasibility in supporting stem cell attachment and differentiation. However, the applicability is significantly hampered by evidence of nanotoxic effects on multiple cell types. Thus, an emergent drive for an innovative carbonaceous biomaterial calls for a safer platform with comparable advantageous characteristics. Here, we showed for the first time, a natural coal-based activated charcoal (AC) composite biosubstrate can support and promote neuronal differentiation in hESCs. The bio-friendly AC composite biomatrices resulted in more matured neuron-like cells. Both of axonal length and density were at least twice as long and abundant, respectively, when compared with control groups. A functional assay demonstrated that the derived neuron-like cells responded to depolarization-dependent synaptic recycling and may contain active synapses. In addition, the AC composite substrate can serve to concentrate growth factors and cell adhesion proteins, further encouraging attachment and hESC differentiation. Moreover, the AC composite biomaterial can potentially be economically manufactured as implantable three-dimensional bioscaffolds, facilitating the regeneration of damaged neural and other tissues.

Human stem cell neuronal differentiation on silk-carbon nanotube composite.

Human embryonic stem cells [hESCs] are able to differentiate into specific lineages corresponding to regulated spatial and temporal signals. This unique attribute holds great promise for regenerative medicine and cell-based therapy for many human diseases such as spinal cord injury [SCI] and multiple sclerosis [MS]. Carbon nanotubes [CNTs] have been successfully used to promote neuronal differentiation, and silk has been widely applied in tissue engineering. This study aims to build silk-CNT composite scaffolds for improved neuron differentiation efficiency from hESCs.Two neuronal markers (ß-III tubulin and nestin) were utilized to determine the hESC neuronal lineage differentiation. In addition, axonal lengths were measured to evaluate the progress of neuronal development. The results demonstrated that cells on silk-CNT scaffolds have a higher ß-III tubulin and nestin expression, suggesting augmented neuronal differentiation. In addition, longer axons with higher density were found to associate with silk-CNT scaffolds.Our silk-CNT-based composite scaffolds can promote neuronal differentiation of hESCs. The silk-CNT composite scaffolds developed here can serve as efficient supporting matrices for stem cell-derived neuronal transplants, offering a promising opportunity for nerve repair treatments for SCI and MS patients.

Effects of engineered nanoparticles on the assembly of exopolymeric substances from phytoplankton.

The unique properties of engineered nanoparticles (ENs) that make their industrial applications so attractive simultaneously raise questions regarding their environmental safety. ENs exhibit behaviors different from bulk materials with identical chemical compositions. Though the nanotoxicity of ENs has been studied intensively, their unintended environmental impacts remain largely unknown. Herein we report experimental results of EN interactions with exopolymeric substances (EPS) from three marine phytoplankton species: Amphora sp., Ankistrodesmus angustus and Phaeodactylum tricornutum. EPS are polysaccharide-rich anionic colloid polymers released by various microorganisms that can assemble into microgels, possibly by means of hydrophobic and ionic mechanisms. Polystyrene nanoparticles (23 nm) were used in our study as model ENs. The effects of ENs on EPS assembly were monitored with dynamic laser scattering (DLS). We found that ENs can induce significant acceleration in Amphora sp. EPS assembly; after 72 hours EN-EPS aggregation reached equilibrium, forming microscopic gels of ∼4-6 µm in size. In contrast, ENs only cause moderate assembly kinetic acceleration for A. angustus and P. tricornutum EPS samples. Our results indicate that the effects of ENs on EPS assembly kinetics mainly depend on the hydrophobic interactions of ENs with EPS polymers. The cycling mechanism of EPS is complex. Nonetheless, the change of EPS assembly kinetics induced by ENs can be considered as one potential disturbance to the marine carbon cycle.

Mucin secretion induced by titanium dioxide nanoparticles.

Nanoparticle (NP) exposure has been closely associated with the exacerbation and pathophysiology of many respiratory diseases such as Chronic Obstructive Pulmonary Disease (COPD) and asthma. Mucus hypersecretion and accumulation in the airway are major clinical manifestations commonly found in these diseases. Among a broad spectrum of NPs, titanium dioxide (TiO(2)), one of the PM10 components, is widely utilized in the nanoindustry for manufacturing and processing of various commercial products. Although TiO(2) NPs have been shown to induce cellular nanotoxicity and emphysema-like symptoms, whether TiO(2) NPs can directly induce mucus secretion from airway cells is currently unknown. Herein, we showed that TiO(2) NPs (<75 nm) can directly stimulate mucin secretion from human bronchial ChaGo-K1 epithelial cells via a Ca(2+) signaling mediated pathway. The amount of mucin secreted was quantified with enzyme-linked lectin assay (ELLA). The corresponding changes in cytosolic Ca(2+) concentration were monitored with Rhod-2, a fluorescent Ca(2+) dye. We found that TiO(2) NP-evoked mucin secretion was a function of increasing intracellular Ca(2+) concentration resulting from an extracellular Ca(2+) influx via membrane Ca(2+) channels and cytosolic ER Ca(2+) release. The calcium-induced calcium release (CICR) mechanism played a major role in further amplifying the intracellular Ca(2+) signal and in sustaining a cytosolic Ca(2+) increase. This study provides a potential mechanistic link between airborne NPs and the pathoetiology of pulmonary diseases involving mucus hypersecretion.

Functionalized positive nanoparticles reduce mucin swelling and dispersion.

Multi-functionalized nanoparticles (NPs) have been extensively investigated for their potential in household and commercial products, and biomedical applications. Previous reports have confirmed the cellular nanotoxicity and adverse inflammatory effects on pulmonary systems induced by NPs. However, possible health hazards resulting from mucus rheological disturbances induced by NPs are underexplored. Accumulation of viscous, poorly dispersed, and less transportable mucus leading to improper mucus rheology and dysfunctional mucociliary clearance are typically found to associate with many respiratory diseases such as asthma, cystic fibrosis (CF), and COPD (Chronic Obstructive Pulmonary Disease). Whether functionalized NPs can alter mucus rheology and its operational mechanisms have not been resolved. Herein, we report that positively charged functionalized NPs can hinder mucin gel hydration and effectively induce mucin aggregation. The positively charged NPs can significantly reduce the rate of mucin matrix swelling by a maximum of 7.5 folds. These NPs significantly increase the size of aggregated mucin by approximately 30 times within 24 hrs. EGTA chelation of indigenous mucin crosslinkers (Ca(2+) ions) was unable to effectively disperse NP-induced aggregated mucins. Our results have demonstrated that positively charged functionalized NPs can impede mucin gel swelling by crosslinking the matrix. This report also highlights the unexpected health risk of NP-induced change in mucus rheological properties resulting in possible mucociliary transport impairment on epithelial mucosa and related health problems. In addition, our data can serve as a prospective guideline for designing nanocarriers for airway drug delivery applications.

A new role for bicarbonate in mucus formation.

The impact of small anions on the physical properties of gel-forming mucin has been almost overlooked relative to that of cations. Recently, based on the coincident abnormalities in HCO(3)(-) secretion and abnormal mucus formed in the hereditary disease cystic fibrosis (CF), HCO(3)(-) was hypothesized to be critical in the formation of normal mucus by virtue of its ability to sequester Ca(2+) from condensed mucins being discharged from cells. However, direct evidence of the impact of HCO(3)(-) on mucus properties is lacking. Herein, we demonstrate for the first time that mucin diffusivity (∼1/viscosity) increases as a function of [HCO(3)(-)]. Direct measurements of exocytosed mucin-swelling kinetics from airway cells showed that mucin diffusivity increases by ∼300% with 20 mM extracellular HCO(3)(-) concentration. Supporting data indicate that HCO(3)(-) reduces free Ca(2+) concentration and decreases the amount of Ca(2+) that remains associated with mucins. The results demonstrate that HCO(3)(-) enhances mucin swelling and hydration by reducing Ca(2+) cross-linking in mucins, thereby decreasing its viscosity and likely increasing its transportability. In addition, HCO(3)(-) can function as a Ca(2+) chelator like EGTA to disperse mucin aggregates. This study indicates that poor HCO(3)(-) availability in CF may explain why secreted mucus remains aggregated and more viscous in affected organs. These insights bear on not only the fundamental pathogenesis in CF, but also on the process of gel mucus formation and release in general.