High-Resolution Patterned Cellular Constructs by Droplet-Based 3D Printing.

Bioprinting is an emerging technique for the fabrication of living tissues that allows cells to be arranged in predetermined three-dimensional (3D) architectures. However, to date, there are limited examples of bioprinted constructs containing multiple cell types patterned at high-resolution. Here we present a low-cost process that employs 3D printing of aqueous droplets containing mammalian cells to produce robust, patterned constructs in oil, which were reproducibly transferred to culture medium. Human embryonic kidney (HEK) cells and ovine mesenchymal stem cells (oMSCs) were printed at tissue-relevant densities (10(7) cells mL(-1)) and a high droplet resolution of 1 nL. High-resolution 3D geometries were printed with features of ≤200 μm; these included an arborised cell junction, a diagonal-plane junction and an osteochondral interface. The printed cells showed high viability (90% on average) and HEK cells within the printed structures were shown to proliferate under culture conditions. Significantly, a five-week tissue engineering study demonstrated that printed oMSCs could be differentiated down the chondrogenic lineage to generate cartilage-like structures containing type II collagen

Activation of subventricular zone stem cells after neuronal injury.

The mammalian subventricular zone (SVZ) has garnered a tremendous amount of attention as a potential source of replacement cells for neuronal injury. This zone is highly neurogenic, harbours stem cells and supports long-distance migration. The general pattern of activation includes increased proliferation, neurogenesis and emigration towards the injury. Intrinsic transcription factors and environmental signalling molecules are rapidly being discovered that may facilitate the induction of these cells to mount appropriate therapeutic responses. The extent of SVZ neurogenesis in humans is controversial. However, tantalizing new data suggest that humans are capable of generating increased numbers of neurons after a variety of diseases

High affinity agonist binding to cloned 5-hydroxytryptamine2 receptors is not sensitive to GTP analogs.

Agonists for GTP-binding protein (G protein)-coupled receptors are thought to bind with high affinity to the complex of receptor and G protein. Nonhydrolyzable GTP analogs have been shown to disrupt this complex and reduce the binding affinity for many agonists. Antagonists are thought to bind to the receptor whether or not it is coupled to the G protein, and therefore binding remains unchanged in the presence of GTP analogs. The binding of the serotonin 5-hydroxytryptamine (5-HT)2 receptor agonists serotonin (5-HT) and 4-bromo-2,5-dimethoxyphenylisopropylamine is not affected by the presence of GTP analogs when the cloned 5-HT2 receptor is expressed in the 293 human embryonic kidney cell line. The same receptor expressed in mouse NIH3T3 cells is partially sensitive to GTP analogs. Both cell lines have similar proportions of agonist and antagonist binding sites, and agonist stimulation of both cell lines leads to a robust increase in phosphoinositide hydrolysis. Differences in GTP metabolism in 293 cells is not likely to be the cause of the observed difference in inhibition of agonist binding, because the cloned 5-HT1A serotonin receptor expressed in these cells is sensitive to GTP analogs. The GTP-insensitive agonist binding is best explained by the existence of a G protein-receptor complex in 293 cells that is not sensitive to GTP analogs. Such a G protein-receptor complex may explain the fraction of agonist binding in the brain that is not sensitive to GTP analogs

A subset of clones in the chick telencephalon arranged in rostrocaudal arrays.

BACKGROUND: Different areas of the vertebrate central nervous system appear to follow different rules during development for determining the position of sibling cells. For example, in the chick hindbrain, clones are frequently confined to a single functional unit that derives from a single rhombomere. In contrast, clones in the mammalian cerebral cortex often cross functional boundaries because of the extensive migration of sibling cells in orthogonal directions. We have investigated whether the pattern of clonal distribution in the chick telencephalon is similar to that of the hindbrain or to the more functionally analogous mammalian cerebral cortex. Progenitor cells in the chick telencephalon were marked using a retroviral library encoding alkaline phosphatase and over 10(5) distinct molecular tags. Patterns of dispersion were detected using alkaline phosphatase histochemistry, followed by the recovery and sequencing of the molecular tag. We also analyzed the phenotypes of cells that occurred within the clones. RESULTS: A subset of progenitors gave rise to clones that were found in rostrocaudal arrays resembling tubes. Arrays were restricted in the mediolateral and dorsoventral planes but could span up to 4 mm in the rostrocaudal direction. They were found throughout the telencephalon and a single clone often spanned more than one telencephalic nucleus. Rostrocaudal clones comprised 60% of clones containing five or more cells and contained many different types of neurons, astrocytes, oligodendrocytes, or various combinations of these cell types. CONCLUSIONS: Telencephalic progenitors are multipotent, producing progeny that become distinct cell types. Clonally related cells can migrate rostrocaudally within domains that are restrained in the mediolateral and dorsoventral directions. A subset of rostrocaudal clones resemble those seen in the mammalian cerebral cortex, with respect to the crossing of functional boundaries, but all rostrocaudal clones differ from the cerebral cortical clones in the pattern of spread of sibling cells, with the rostrocaudal clones being more constrained in the mediolateral and dorsoventral directions. A role for lineage in the patterning of the chick forebrain is supported by these observations. In addition, these data suggest a role for cues within the telencephalic marginal zone that serve to guide clones in their rostrocaudal migration

Cortical lesions induce an increase in cell number and PSA-NCAM expression in the subventricular zone of adult rats.

The subventricular zone (SVZ) bordering the lateral ventricle is one of the few regions of adult brain that contains dividing cells. These cells can differentiate into neurons in vivo after migration into the olfactory bulb and in vitro in the presence of appropriate growth factors. Little is known, however, about the fate of these cells in vivo after brain injury in adults. We examined cell number and expression of differentiation markers in the SVZ of adult rats after cortical lesions. Aspiration lesions of the sensorimotor cortex in adult rats induced a transient doubling of the number of cells in the SVZ at the level of the striatum without consistent increases in bromodeoxyuridine-labeled cells. Immunoreactivity to the polysialylated neural cell adhesion molecule, expressed by the majority of cells of the SVZ during development, increased dramatically after lesion. In contrast, immunolabeling for molecules found in mature neurons and glia did not increase in the SVZ after lesion, and immunoreactivity for growth factors that induce differentiation of SVZ cells in vitro decreased or remained undetectable, suggesting that lack of appropriate growth factor expression may contribute to the lack of differentiation of the newly accumulated cells in vivo. The data reveal that cells of the SVZ are capable of plasticity in the adult rat after brain injury in vivo and that the newly accumulated cells retain characteristics seen during development

The dispersion of clonally related cells in the developing chick telencephalon.

Lineage analysis in the chick telencephalon was carried out using a library of retroviral vectors. Clones were analyzed in posthatch day 14-21 animals for the phenotype and final locations of sibling cells. Clones often contained multiple types of neurons and glia. Clones of more than four cells almost always crossed functional boundaries. They were dispersed primarily along the rostrocaudal axis or in multiple directions, e.g., along the rostrocaudal and mediolateral axes. In order to begin to understand how the final patterns of dispersion were reached, embryonic tissue was examined. Radial migration, apparently supported by radial glial cells, occurred within the proliferative zones in all clones. In contrast to the migration of cells in the mammalian telencephalon, no tangential migration within the proliferative zones was observed at any age examined. However, beginning at embryonic day 4.5, tangential migration in the mantle zone in multiple directions was observed among the majority of clones. This type of migration occurred as soon as a mantle zone became apparent. It appeared that the tangential migration was not along radial glial processes. As in the mammalian telencephalon and chick diencephalon, dispersion among clonally related cells in the chick telencephalon is frequent, is extensive, and results from tangential migration in a variety of directions

Expression of molecules associated with neuronal plasticity in the striatum after aspiration and thermocoagulatory lesions of the cerebral cortex in adult rats.

Like the hippocampus, the striatum receives excitatory afferents from the cerebral cortex but, in the case of the striatum, very little is known about the molecular events associated with plasticity after lesions of this pathway. Using immunohistochemical techniques, we have examined the effects of cortical lesions induced either by aspiration of the frontoparietal cortex or by thermocoagulation of pial blood vessels on axonal and glial molecules associated with neuronal plasticity in the striatum. The growth associated protein GAP-43, a molecule present in axons and growth cones, decreased in the dorsolateral striatum after aspiration but not after thermocoagulatory lesions. In contrast, synaptophysin, a marker of synaptic vesicles, remained unchanged in the denervated striatum after both types of lesions. Immunostaining for basic fibroblast growth factor (bFGF) markedly decreased in striatal astrocytes after both lesions, despite an increased staining for glial fibrillary acidic protein (GFAP). The adhesion molecules tenascin, chondroitin sulfate proteoglycans, highly polysialylated neural cell adhesion molecule (PSA-NCAM), and laminin did not change significantly in the gray matter of the dorsolateral striatum after either type of lesion. These effects differed from those observed after partial denervation of the hippocampus and spinal cord, revealing marked regional differences in the response of axonal and glial proteins to afferent lesions. In addition, the results further indicate that cortical lesions have both similar and distinct consequences, depending on the procedure by which the lesions are induced, suggesting that cortical lesions associated with different types of pathology may differentially affect subcortical structures