The directed differentiation of forebrain neuronal types from human embryonic stem cells (hESCs) has not been achieved. Here, we show that hESCs differentiate to telencephalic progenitors with a predominantly dorsal identity in a chemically defined medium without known morphogens. This is attributed to endogenous Wnt signaling, which upregulates the truncated form of GLI3, a repressor of sonic hedgehog (SHH). A high concentration of SHH, or the inhibition of Wnt by dickkopf 1 (DKK1) together with a low concentration of SHH, almost completely converts the primitive dorsal precursors to ventral progenitors, which is partially achieved through both downregulation of the truncated GLI3 and upregulation of full-length GLI3 expression. These dorsal and ventral telencephalic progenitors differentiate to functional glutamatergic and GABAergic neurons, respectively. Thus, although hESCs generate dorsal telencephalic cells, as opposed to ventral progenitors in other vertebrates, in the absence of exogenous morphogens, human cells use a similar molecular mechanism to control the dorsal versus ventral fate. The coordination of Wnt and SHH signaling through GLI3 represents a novel mechanism that regulates ventral-dorsal patterning in the development of forebrain neuronal subtypes.
How a naive human neuroepithelial cell becomes an electrophysiologically active neuron remains unknown. Here, we describe the early physiological development of neurons differentiating from naive human embryonic stem (hES) cells. We found that differentiating neuronal cells progressively decrease their resting membrane potential, gain characteristic Na ϩ and K ϩ currents, and fire mature action potentials by 7 weeks of differentiation. This is similar to the maturation pattern observed in animals, albeit on a greatly expanded time scale. An additional 3 weeks of differentiation resulted in neurons that could fire repetitive trains of action potentials in response to depolarizing current pulses. The onset of spontaneous synaptic activity also occurred after 7 weeks of differentiation, in association with the differentiation of astrocytes within the culture. Cocultures of hES cell-derived neuroepithelial cells with exogenous astrocytes significantly accelerated the onset of synaptic currents but did not alter action potential generation. These findings suggest that the development of membrane characteristics and action potentials depend on the intrinsic maturation of Na ϩ and K ϩ currents, whereas synaptic transmission is enhanced by astrocytes, which may be achieved independently of the maturation of action potentials. Furthermore, we found that although astrocyte-conditioned medium accelerated synaptic protein localization, it did not increase synaptic activity, suggesting a contact-dependant mechanism by which astrocytes augment synaptic activity. These results lay the foundation for future studies examining the functional development of human neurons and provide support for the potential application of human cells in restorative neuronal therapies.
A number of polydimethysiloxane (PDMS) bonding techniques have been reported in the literature over the last several years as the focus on multilayer PDMS microfluidic devices has increased. Oxygen plasma bonding, despite cost, additional fabrication time and inconsistent bonding results, has remained a widely used method for bonding PDMS layers. A comparative study of four rapid, inexpensive alternative PDMS-PDMS bonding approaches was undertaken to determine relative bond strength. These include corona discharge, partial curing, cross-linker variation and uncured PDMS adhesive. Partial curing and uncured PDMS adhesive demonstrated a considerable improvement in bond strength and consistency by retaining average bond strengths of over 600 kPa, which was more than double the average bond strength of oxygen plasma. A description of each technique and their performance relative to oxygen plasma bonding is included.
Due to the lack of development in the area of sample preparation, few complete lab-on-a-chip systems have appeared in recent years that can deal with raw samples. Cell lysis and nucleic acid extraction systems are sufficiently complex even before adding the complexity of an analysis system. In this review, a variety of microfluidic sample preparation methods are discussed and evaluated. Microsystems for cell lysis are discussed by grouping them into categories based on their lysis mechanisms: mechanical, chemical, thermal or electrical. We classify the nucleic acid purification techniques according to the mechanism that links nucleic acids to substrates: silica-based surface affinity, electrostatic interaction, nanoporous membrane filtration, and functionalized microparticles. The techniques for microfluidic cell lysis and nucleic acid purification are compared based on the ease of microfabrication and integration, and sample flexibility. These assessments can help us determine the appropriate sample preparation technique for generating a true lab-on-a-chip.
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