Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations

Background  

The present work was performed to investigate the ability of two different embryonic stem (ES) cell-derived neural precursor populations to generate functional neuronal networks in vitro. The first ES cell-derived neural precursor population was cultivated as free-floating neural aggregates which are known to form a developmental niche comprising different types of neural cells, including neural precursor cells (NPCs), progenitor cells and even further matured cells. This niche provides by itself a variety of different growth factors and extracellular matrix proteins that influence the proliferation and differentiation of neural precursor and progenitor cells. The second population was cultivated adherently in monolayer cultures to control most stringently the extracellular environment. This population comprises highly homogeneous NPCs which are supposed to represent an attractive way to provide well-defined neuronal progeny. However, the ability of these different ES cell-derived immature neural cell populations to generate functional neuronal networks has not been assessed so far.     

Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations

Background

Minocycline, a second-generation tetracycline with anti-inflammatory and anti-apoptotic properties, has been shown to promote therapeutic benefits in experimental stroke. However, equally compelling evidence demonstrates that the drug exerts variable and even detrimental effects in many neurological disease models. Assessment of the mechanism underlying minocycline neuroprotection should clarify the drug's clinical value in acute stroke setting.

Results

Here, we demonstrate that minocycline attenuates both in vitro (oxygen glucose deprivation) and in vivo (middle cerebral artery occlusion) experimentally induced ischemic deficits by direct inhibition of apoptotic-like neuronal cell death involving the anti-apoptotic Bcl-2/cytochrome c pathway. Such anti-apoptotic effect of minocycline is seen in neurons, but not apparent in astrocytes. Our data further indicate that the neuroprotection is dose-dependent, in that only low dose minocycline inhibits neuronal cell death cascades at the acute stroke phase, whereas the high dose exacerbates the ischemic injury.

Conclusion

The present study advises our community to proceed with caution to use the minimally invasive intravenous delivery of low dose minocycline in order to afford neuroprotection that is safe for stroke.     

Directed neuronal differentiation of human embryonic stem cells

Background

We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs) to neural precursors and neurons.HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4⁺/SSEA-4^- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII).

Results

A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7–10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons.

Conclusion

This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4⁺ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.

     

c-MycERTAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain

Background

To date, functional imaging studies of treatment-induced recovery from chronic aphasia only assessed short-term treatment effects after intensive language training. In the present study, we show with functional magnetic resonance imaging (fMRI), that different brain regions may be involved in immediate versus long-term success of intensive language training in chronic post-stroke aphasia patients.

Results

Eight patients were trained daily for three hours over a period of two weeks in naming of concrete objects. Prior to, immediately after, and eight months after training, patients overtly named trained and untrained objects during event-related fMRI. On average the patients improved from zero (at baseline) to 64.4% correct naming responses immediately after training, and treatment success remained highly stable at follow-up. Regression analyses showed that the degree of short-term treatment success was predicted by increased activity (compared to the pretraining scan) bilaterally in the hippocampal formation, the right precuneus and cingulate gyrus, and bilaterally in the fusiform gyri. A different picture emerged for long-term training success, which was best predicted by activity increases in the right-sided Wernicke's homologue and to a lesser degree in perilesional temporal areas.

Conclusion

The results show for the first time that treatment-induced language recovery in the chronic stage after stroke is a dynamic process. Initially, brain regions involved in memory encoding, attention, and multimodal integration mediated treatment success. In contrast, long-term treatment success was predicted mainly by activity increases in the so-called 'classical' language regions. The results suggest that besides perilesional and homologue language-associated regions, functional integrity of domain-unspecific memory structures may be a prerequisite for successful (intensive) language interventions.     

Differential orientation effect in the neural response to interacting biological motion of two agents

Background  

A recent behavioral study demonstrated that the meaningful interaction of two agents enhances the detection sensitivity of biological motion (BM), however, it remains unclear when and how the 'interaction' information of two agents is represented in our neural system. To clarify this point, we used magnetoencephalography and introduced a novel experimental technique to extract a neuromagnetic response relating to two-agent BM perception. We then investigated how this response was modulated by the interaction of two agents. In the present experiment, we presented two kinds of visual stimuli (interacting and non-interacting BM) with two orientations (upright and inverted).     

Differentiation of human bone marrow stem cells into cells with a neural phenotype: diverse effects of two specific treatments

Background  

It has recently been demonstrated that the fate of adult cells is not restricted to their tissues of origin. In particular, it has been shown that bone marrow stem cells can give rise to cells of different tissues, including neural cells, hepatocytes and myocytes, expanding their differentiation potential.     

Laminin enhances the growth of human neural stem cells in defined culture media

Background  

Human neural stem cells (hNSC) have the potential to provide novel cell-based therapies for neurodegenerative conditions such as multiple sclerosis and Parkinson's disease. In order to realise this goal, protocols need to be developed that allow for large quantities of hNSC to be cultured efficiently. As such, it is important to identify factors which enhance the growth of hNSC. In vivo, stem cells reside in distinct microenvironments or niches that are responsible for the maintenance of stem cell populations. A common feature of niches is the presence of the extracellular matrix molecule, laminin. Therefore, this study investigated the effect of exogenous laminin on hNSC growth.     

Differential responses of cancer cell lines to non-thermal plasma from dielectric barrier discharge

The differential responses of six cancer cell lines after treatment of non-thermal plasma from dielectric barrier discharge (DBD) were studied. Two plasma exposure methods including cell suspension exposure and media only exposure were used, and the effects were examined in six cancer cell lines including KB, MCF-7, HeLa, H460, SNU-80 and T98G from different human organs. The result indicates that both methods affected six cell lines similarly in proliferation, mitochondria activity, and apoptosis related gene expression, which implies that the cell culture media have significant role in plasma–cell interaction. H460 showed a significant reduction in cell number after plasma exposure, whereas MCF-7 showed less reduction. Mitochondria activity of all the cancer cell lines reduced following exposure and incubation times except MCF-7. The mRNA expression of Bax, the apoptosis related gene, was highly enhanced in KB, HeLa, MCF-7 and SNU-80, while none was detected in T98G. All six cancer cell lines showed unexpectedly low mRNA expression of tumor suppressor gene p53. These differential responses of six cell lines suggest that non-thermal plasma should activate different pathways in each cancer cell line. Further researches on tissue-specific responses to plasma treatment should be conducted, comparing their responses with normal tissue–cell responses.     

The hematopoietic factor GM-CSF (Granulocyte-macrophage colony-stimulating factor) promotes neuronal differentiation of adult neural stem cells in vitro

Background  

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor involved in the generation of granulocytes, macrophages, and dendritic cells from hematopoietic progenitor cells. We have recently demonstrated that GM-CSF has anti-apoptotic functions on neurons, and is neuroprotective in animal stroke models.     

Differential production of superoxide by neuronal mitochondria

Background  

Mitochondrial DNA (mtDNA) mutations, which are present in all mitochondria-containing cells, paradoxically cause tissue-specific disease. For example, Leber's hereditary optic neuropathy (LHON) results from one of three point mutations mtDNA coding for complex I components, but is only manifested in retinal ganglion cells (RGCs), a central neuron contained within the retina. Given that RGCs use superoxide for intracellular signaling after axotomy, and that LHON mutations increase superoxide levels in non-RGC transmitochondrial cybrids, we hypothesized that RGCs regulate superoxide levels differently than other neuronal cells. To study this, we compared superoxide production and mitochondrial electron transport chain (METC) components in isolated RGC mitochondria to mitochondria isolated from cerebral cortex and neuroblastoma SK-N-AS cells.     

Period 2 regulates neural stem/progenitor cell proliferation in the adult hippocampus

Background  

Newborn granule neurons are generated from proliferating neural stem/progenitor cells and integrated into mature synaptic networks in the adult dentate gyrus of the hippocampus. Since light/dark variations of the mitotic index and DNA synthesis occur in many tissues, we wanted to unravel the role of the clock-controlled Period2 gene (mPer2) in timing cell cycle kinetics and neurogenesis in the adult DG.     

The effect of ultrasonic irradiation on doxorubicin-induced cytotoxicity in three human bladder cancer cell lines

Although ultrasonic irradiation has been proven to increase membrane permeability and enhance chemotherapeutic cytotoxicity in a number of cell lines, this effect has never been demonstrated in bladder cancer cells. Bladder cancer may offer a unique setting for ultrasound enhancement of chemotherapy, since intravesicular rather than intravenous administration of chemotherapy is used in superficial cases. The aim of this study was to investigate whether a non-toxic dose of ultrasound could increase membrane permeability, and potentiate the cytotoxicity of doxorubicin to three human bladder carcinoma cell lines (TCC-SUP, T24, and RT4) in vitro. An EuTDA-Efflux assay, which measures the amount of a chemical that is allowed to seep out of labeled cells, was used to analyze membrane permeability, and an MTS assay, which directly measures cell viability, was used to determine the effect of chemotherapy on cells after they were treated with a variety of doxorubicin concentrations and ultrasonic exposures. Ultrasound treatment for 5 min and 10 min at an intensity of approximately 0.3 W/cm² resulted in a significant increase in EuTDA efflux in all three cell lines. However, no ultrasonic enhancement of doxorubicin growth inhibition in these human bladder carcinoma cells was observed. This suggests that either ultrasound does not increase doxorubicin uptake by the cell or that doxorubicin uptake is increased but in insufficient amounts to affect growth inhibition. Further investigation should focus on explaining these results.     

Effects of hematin and Mössbauer gamma-radiation on human osteosarcoma cell lines

Summary In the present work we report an investigation on cultures of the human osteosarcoma cell line ?MG-63? and the human osteoblastic cell line ?HOBIT?. We have examined the combined effects of hematin and 14.4keV gamma-radiation from a M?ssbauer source. Preliminary results seem to confirm the trend already observed for the bone marrow system. Different degrees of growth inhibition were observed when hematin alone and hematin plus gamma-rays were administered to the cultures. It was previously found that hematin has no toxic effects on normal cells up to a concentration of 10⁻⁴ M, but the same concentration produces various degrees of inhibition on cultures of tumor cells. While no significant effect could be attributed to irradiation alone, hematin plus irradiations show a larger inhibition than that expected for pure additive effects. Paper presented at ICAME-95, Rimini, 10–16 September 1995.     

Healthy and unhealthy red blood cell detection in human blood smears using neural networks

One of the most common diseases that affect human red blood cells (RBCs) is anaemia. To diagnose anaemia, the following methods are typically employed: an identification process that is based on measuring the level of haemoglobin and the classification of RBCs based on a microscopic examination in blood smears. This paper presents a proposed algorithm for detecting and counting three types of anaemia-infected red blood cells in a microscopic coloured image using circular Hough transform and morphological tools. Anaemia cells include sickle, elliptocytosis, microsite cells and cells with unknown shapes. Additionally, the resulting data from the detection process have been analysed by a prevalent data analysis technique: the neural network. The experimental results for this model have demonstrated high accuracy for analysing healthy/unhealthy cells. This algorithm has achieved a maximum detection of approximately 97.8% of all cells in 21 microscopic images. Effectiveness rates of 100%, 98%, 100%, and 99.3% have been achieved using neural networks for sickle cells, elliptocytosis cells, microsite cells and cells with unknown shapes, respectively.     

Cerebrospinal fluid promotes survival and astroglial differentiation of adult human neural progenitor cells but inhibits proliferation and neuronal differentiation

Background  

Neural stem cells (NSCs) are a promising source for cell replacement therapies for neurological diseases. Growing evidence suggests an important role of cerebrospinal fluid (CSF) not only on neuroectodermal cells during brain development but also on the survival, proliferation and fate specification of NSCs in the adult brain. Existing in vitro studies focused on embryonic cell lines and embryonic CSF. We therefore studied the effects of adult human leptomeningeal CSF on the behaviour of adult human NSCs (ahNSCs).     

Evidence for two discrete sources of 2f1-f2 distortion-product otoacoustic emission in rabbit. II: Differential physiological vulnerability.

In a previous report, it was shown that, in normal rabbit ears, the amplitude and phase of 2f1-f2 distortion-product otoacoustic emissions (DPOAEs) elicited by low-level (< 60-70 dB SPL) stimuli display a differential dependence on stimulus parameters to those evoked by high-level (> 60-70 dB SPL) stimuli, indicating differences in the underlying generation mechanisms. In the present study, the physiological vulnerability of DPOAEs in each of the two 2f1-f2 DPOAE-response regions identified on the basis of differential parametric properties, was characterized. Thus emissions evoked using stimulus levels from 45-75 dB SPL were measured over time upon: (1) induction of lethal anoxia, (2) acute injection of ethacrynic acid, and (3) acute injection of ethacrynic acid 2 h after a single administration of gentamicin. The DPOAEs evoked by low-level stimuli (45 dB SPL) were abolished within 3-4 min of induction of anoxia, whereas DPOAEs evoked by high-level stimuli (75 dB SPL) were unchanged in this period. The high-level emissions decreased with a complex time course postmortem, and demonstrated behaviors, including evidence of susceptibility to fatigue, suggesting a dependence upon a cochlear energy supply. Low-level DPOAEs could be temporarily abolished, with complete recovery, by an acute administration of ethacrynic acid that had little effect on high-level DPOAEs. Treatment with the gentamicin and ethacrynic-acid combination, which would be expected to produce widespread hair-cell damage, eliminated low-level DPOAEs, and greatly reduced high-level emissions. In combination with previously published data, these findings strongly suggest that low- and high-level 2f1-f2 DPOAEs arise from discrete sources. The data are consistent with the proposal that the low-level DPOAE source is an active, micromechanical process, but suggest that the proposed origin of high-level DPOAEs exclusively in the passive macromechanics of the cochlear partition may be incorrect. The elimination of both low- and high-level DPOAEs revealed the presence of a third, residual 2f1-f2 DPOAE component, approximately 75-80 dB below the stimulus-tone levels, that may reflect the true passive-distortion response of the cochlea.     

Biological effects of ultrasound exposure on adriamycin-resistant and cisplatin-resistant human ovarian carcinoma cell lines in vitro

Human ovarian cancer cell lines, SKOV3 and its adriamycin-resistant substrain SKOV3/ADR and COC1 and its cisplatin-resistant substrain COC1/DDP, were subjected to acoustic exposure. The critical levels (LC), which resulted in no immediate cell killing, were determined in four cell lines, respectively. LC were the same in four cell lines. After being insonated by LC, cell proliferation and clone forming of SKOV3/ADR were suppressed but those of SKOV3 were not affected (1); cell reproduction of COC1 was triggered but that of COC1/DDP was not influenced (2); flow cytometry detected sub-G1 peaks in SKOV3/ADR and COC1/DDP (3). These findings suggested that there were differences in the responses to ultrasound exposure between chemosensitive and chemoresistant human ovarian cancer cells.