Presence Of Astrocyte-conditioned Medium Research Articles

Identification of Neural Stem Cells from Postnatal Mouse Auditory Cortex In Vitro.

Auditory signals are processed in multiple central nervous system structures, including the auditory cortex (AC). Development of stem cell biology provides the opportunity to identify neural stem cells (NSCs) in the central nervous system. However, it is unclear whether NSCs exist in the AC. The aim of this study is to determine the existence of NSCs in the postnatal mouse AC. To accomplish this aim, postnatal mouse AC tissues were dissected and dissociated into singular cells and small cell clumps, which were suspended in the culture medium to observe neurosphere formation. The spheres were examined by quantitative real-time polymerase chain reaction and immunofluorescence to determine expression of NSC genes and proteins. In addition, AC-spheres were cultured in the presence or absence of astrocyte-conditioned medium (ACM) to study neural differentiation. The results show that AC-derived cells were able to proliferate to form neurospheres, which expressed multiple NSC genes and proteins, including SOX2 and NESTIN. AC-derived NSCs (AC-NSCs) differentiated into cells expressing neuronal and glial cell markers. However, the neuronal generation rate is low in the culture medium containing nerve growth factor, ∼8%. To stimulate neuronal generation, AC-NSCs were cultured in the culture medium containing ACM. In the presence of ACM, ∼29% AC-NSCs differentiated into cells expressing neuronal marker class III β-tubulin (TUJ1). It was observed that the length of neurites of AC-NSC-derived neurons in the ACM group was significantly longer than that of the control group. In addition, synaptic protein immunostaining showed significantly higher expression of synaptic proteins in the ACM group. These results suggest that ACM is able to stimulate neuronal differentiation, extension of neurites, and expression of synaptic proteins. Identifying AC-NSCs and determining effects of ACM on NSC differentiation will be important for the auditory research and other neural systems.

Stimulation of synapse formation between stem cell-derived neurons and native brainstem auditory neurons

Integration of stem cell-derived cells into native cellular environment remains a challenge in the field. This study developed novel methods to co-culture neural stem cell-derived spiral ganglion-like neurons (ScNs) and mouse auditory cochlear nucleus (CN) neurons to understand whether ScNs of the peripheral nervous system (PNS) synapse with CN neurons of the central nervous system (CNS). ScNs were obtained from neural stem cells that were derived from transgenic mouse pre-labeled with enhanced green fluorescent protein (EGFP), whereas CN neurons were from postnatal mouse primary cultures. ScNs and CN neurons were co-cultured for 4–6 days in the absence or presence of astrocyte-conditioned medium (ACM). Class III β-tubulin (TUJ1)-expressing connections were found between ScNs and CN neurons. Expression of the synaptic vesicle marker SV2 was significantly increased along connections between ScNs and CN neurons in the presence of ACM. Immunodepletion and knockout studies indicated that thrombospodin-1 played an important role in ACM-exerted synaptogenic effects. Newly-generated synapse-like structures expressed glutamatergic marker VGluT1, pre- and post-synaptic proteins. Synaptic vesicle recycling studies suggested functional synaptic vesicle retrieval. These results reveal that stem cell-derived PNS neurons are able to form functional connections with native CNS neurons, which is critical for stem cell-based neural pathway regeneration.

A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip.

Studies of neonatal neural pathologies and development of appropriate therapeutics are hampered by a lack of relevant in vitro models of neonatal blood-brain barrier (BBB). To establish such a model, we have developed a novel blood-brain barrier on a chip (B3C) that comprises a tissue compartment and vascular channels placed side-by-side mimicking the three-dimensional morphology, size and flow characteristics of microvessels in vivo. Rat brain endothelial cells (RBEC) isolated from neonatal rats were seeded in the vascular channels of B3C and maintained under shear flow conditions, while neonatal rat astrocytes were cultured under static conditions in the tissue compartment of the B3C. RBEC formed continuous endothelial lining with a central lumen along the length of the vascular channels of B3C and exhibited tight junction formation, as measured by the expression of zonula occludens-1 (ZO-1). ZO-1 expression significantly increased with shear flow in the vascular channels and with the presence of astrocyte conditioned medium (ACM) or astrocytes cultured in the tissue compartment. Consistent with in vivo BBB, B3C allowed endfeet-like astrocyte-endothelial cell interactions through a porous interface that separates the tissue compartment containing cultured astrocytes from the cultured RBEC in the vascular channels. The permeability of fluorescent 40 kDa dextran from vascular channel to the tissue compartment significantly decreased when RBEC were cultured in the presence of astrocytes or ACM (from 41.0±0.9 x 10−6 cm/s to 2.9±1.0 x 10−6 cm/s or 1.1±0.4 x 10−6 cm/s, respectively). Measurement of electrical resistance in B3C further supports that the addition of ACM significantly improves the barrier function in neonatal RBEC. Moreover, B3C exhibits significantly improved barrier characteristics compared to the transwell model and B3C permeability was not significantly different from the in vivo BBB permeability in neonatal rats. In summary, we developed a first dynamic in vitro neonatal BBB on a chip (B3C) that closely mimics the in vivo microenvironment, offers the flexibility of real time analysis, and is suitable for studies of BBB function as well as screening of novel therapeutics.

Transendothelial permeability of chlorpyrifos in RBE4 monolayers is modulated by astrocyte-conditioned medium

The immortalized rat brain endothelium 4 (RBE4) cell line preserves many features of the in vivo brain endothelium. It has been used as an in vitro model of the blood–brain barrier (BBB). Astrocyte–endothelial cell interactions are crucial for maintenance of BBB characteristics. The present study investigated morphological and permeability properties of the RBE4 cell line. Immunohistochemical studies showed positive staining in RBE4 cells for E-cadherin, a Ca 2+-dependent cell–cell adhesion molecule. Western blot immunoassay showed that RBE4 cells consistently express E-cadherin and that its expression significantly increased ( P<0.001) in the presence of astrocyte-conditioned medium (ACM). The transendothelial permeability of chlorpyrifos, an organophosphorus insecticide, was significantly decreased ( P<0.001) when the RBE4 cells were grown in ACM compared with control medium. Additional studies were carried out to determine whether chlorpyrifos is a substrate for the multidrug resistance protein, P-glycoprotein (P-gp). No significant change in chlorpyrifos transendothelial permeability was noted in the presence of verapamil, a P-gp blocker. Thus, in this system, chlorpyrifos is not a substrate for P-gp. This work demonstrates that with additional refinements the RBE4 monolayers might serve as a useful in vitro model for the study of BBB permeability and modulation by astrocyte-derived soluble factors.

ATP and adenosine induce ramification of microglia in vitro

Microglial cells in the healthy adult brain possess a characteristic ramified morphology with multiple branched processes, small somata and down-regulated inflammatory properties. In contrast, microglial cells isolated from new-born rat brain inevitably show a non-ramified amoeboid phenotype, which is observed in vivo after pathologic activation or during development. To identify factors that control microglial morphology we investigated the effects of purines alone or in combination with astrocyte-conditioned medium (ACM). Under optimized culture conditions postnatal rat microglial cells developed an amoeboid to ovoid phenotype. Addition of 0.6–1 mM ATP or adenosine induced the outgrowth of numerous processes after 2–3 days that could be observed also in the presence of ACM as previously reported. Culture in ACM plus ATP or adenosine yielded an optimized ramified phenotype. ATP or adenosine, but not ACM alone, also prevented the formation of a flat, amoeboid morphology induced by lipopolysaccharide (LPS); however, at 0.6–1 mM they did not reduce the initial LPS-induced activation of the transcription factor NF- κB. By using specific agonists or antagonists the morphological transformations could not be confined to a distinct purinoreceptor subtype, but appeared to be mediated by long-term presence of adenosine in the medium to which phosphorylated purines were rapidly hydrolyzed by microglial cells. Since ACM did not contain sufficient concentrations of ATP or adenosine, purines are not the only ramification-inducing factors present in ACM; however, they are a valuable tool to induce microglial ramification in vitro.

Astrocytes protect against X-ray-induced neuronal toxicity in vitro

The role of neuronal damage in the radiation-induced CNS injury resulting from brain tumor therapy remains poorly understood. To begin to define the radioresponse of neurons, the survival of rat cortical neuron cultures was investigated. Neuronal survival was reduced by approximately 40% 24-48 h after irradiation with 3.5 Gy. The addition of the free radical scavenger NAC after irradiation increased neuronal survival. Neurons were also significantly less sensitive to radiation in co-cultures with astrocytes or in the presence of astrocyte-conditioned medium. Medium conditioned on astrocytes was found to acquire significant free radical scavenging capability. However, this antioxidant property does not appear to be responsible for neuronal radioprotection. The ability of astrocytes to reduce radiation-induced neuronal toxicity appears to be mediated by a soluble protein(s) of mol. wt > 10 kDa.

Regulation of production by primary cultures of rat forebrain astrocytes of a trophic factor important for the development of cholinergic neurons

The possibility was examined for the production of a trophic factor by astrocytes important for the biochemical development of cholinergic neurons. The activity of choline acetyltransferase (ChAT) was used as a marker to study the maturation of cholinergic neurons, while the metabolic state of astrocytes was monitored in terms of glutamine synthetase activity. When the dissociated cells, derived from the septal-diagonal band region of embryonic rat brain, were cultured either on a preformed layer of highly enriched astrocytes or in the presence of astrocyte-conditioned medium, a marked increase was observed in the expression of ChAT activity. Similar effect was not noticed when cerebellar granule cells replaced the astrocytes. The production of cholinergic factor was decreased when the astroglial cells were induced to proliferate by epidermal growth factor, whereas its production was increased when quiescent astrocytes were treated with a low dose of cytosine arabinoside. The results would indicate that a trophic factor important for the development of cholinergic cells is produced by astrocytes, and that exogenously added agents influencing astroglial metabolism have a marked regulatory effect on the production of this trophic factor.