Downregulation of CDC2 upon terminal differentiation of neurons.

The terminal differentiation of neurons occurs as precisely timed waves, with specific neuronal types differentiating in defined sequences. The precision of neuronal differentiation in the central nervous system offers an unusual opportunity to study terminal differentiation in vivo. The p34cdc2 kinase complex and the anti-oncogenes p53 and RB are central in the regulatory network that controls cell proliferation. We found high levels of expression of CDC2 mRNA and protein in proliferating neuronal precursor cells. The expression of both CDC2 and cyclin A was dramatically downregulated upon terminal differentiation of neurons in vivo and in a neuronal precursor cell line, ST15A. p53 mRNA expression was also downregulated but to a lesser extent; RB mRNA levels were unchanged during neuronal differentiation. Immunohistochemistry showed that p34cdc2 was expressed not only in the neuronal precursors of the cerebellar external granule layer but also in the early differentiating granule neurons. The expression of p34cdc2 in early neurons suggests a function for this enzyme in the events that occur soon after proliferation ceases. On the basis of the results reported here and other recent findings, we propose a model in which terminal differentiation is achieved by a switch in the neuronal precursors from p34cdc2-based proliferation to a differentiated state controlled by p34cdc2-related kinases.     

Regulation of miR-34 Family in Neuronal Development

Differentiation of neural stem cells (NSC’s) to mature and functional neurons requires coordinated expression of mRNA, microRNAs (miRNAs) and regulatory proteins. Our earlier unbiased miRNA profiling studies have identified miR-200, miR-34 and miR-221/222 as maximally up-regulated miRNA families in differentiating PC12 cells and demonstrated the capability of miR-200 family in inducing neuronal differentiation (J. Neurochem, 2015, 133, 640–652). In present study, we have investigated role of miR-34 family in neuronal differentiation and identified P53 as mediator of nerve growth factor (NGF) induced miR-34a expression in differentiating PC12 cells. Our studies have shown that NGF induced miR-34a, arrests proliferating PC12 cells to G1 phase, which is pre-requisite for neuronal differentiation. Our studies have also shown that increased expression of miR-34a controls the P53 level in differentiated PC12 cells in feedback inhibition manner, which probably prevents differentiated cells from P53 induced apoptosis. Expression profiling of miR-34 family in different neuronal, non-neuronal and developing cells have identified differentiated and aged brain cells as richest source of miR-34, which also indicates that higher expression of miR-34 family helps in maintaining the mature neurons in non-proliferative stage. In conclusion, our studies have shown that miR-34 is brain enriched miRNA family, which up-regulates with neuronal maturation and brain ageing and co-operative regulation of P53 and miR-34a helps in neuronal differentiation by arresting cells in G1 phase.     

Characteristics of human embryonic neuronal cells procured by non-enzyme method

Isolation and culturing of human neuronal progenitor cells is of significant value for both fundamental research and therapeutic purposes. In this work, human embryonic neuronal cells were characterized as a heterogeneous population of progenitor cells with various differentiation potentials. During in vitro culturing the cells are capable of re-inoculating, proliferating, differentiating and migrating. While differentiating, these cells form neurons and glial cells. The present research demonstrates that depending upon the culturing conditions the embryonic neuronal cells may either form floating aggregates (incubation with embryonic serum), attach (incubation without serum), proliferate, or form neurospheres. Besides, peculiarities of aggregate differentiation during their incubation under various media are described.     

Reduced expression of MECP2 affects cell commitment and maintenance in neurons by triggering senescence: new perspective for Rett syndrome

MECP2 protein binds preferentially to methylated CpGs and regulates gene expression by causing changes in chromatin structure. The mechanism by which impaired MECP2 activity can induce pathological abnormalities in the nervous system of patients with Rett syndrome (RTT) is not clearly understood. To gain further insight into the role of MECP2 in human neurogenesis, we compared the neural differentiation process in mesenchymal stem cells (MSCs) obtained from a RTT patient and from healthy donors. We further analyzed neural differentiation in a human neuroblastoma cell line carrying a partially silenced MECP2 gene. Senescence and reduced expression of neural markers were observed in proliferating and differentiating MSCs from the RTT patient, which suggests that impaired activity of MECP2 protein may impair neural differentiation, as observed in RTT patients. Next, we used an inducible expression system to silence MECP2 in neuroblastoma cells before and after the induction of neural differentiation via retinoic acid treatment. This approach was used to test whether MECP2 inactivation affected the cell fate of neural progenitors and/or neuronal differentiation and maintenance. Overall, our data suggest that neural cell fate and neuronal maintenance may be perturbed by senescence triggered by impaired MECP2 activity either before or after neural differentiation.     

TGF-beta and retinoic acid: regulators of growth and modifiers of differentiation in human epidermal cells.

In the epidermis of skin, a fine balance exists between proliferating progenitor cells and terminally differentiating cells. We examined the effects of TGF-beta s and retinoic acid (RA) on controlling this balance in normal and malignant human epidermal keratinocytes cultured under conditions where most morphological and biochemical features of epidermis in vivo are retained. Our results revealed marked and pleiotropic effects of both TGF-beta and RA on keratinocytes. In contrast to retinoids, TGF-beta s acted on mitotically active basal cells to retard cell proliferation. Although withdrawal from the cell cycle is a necessary prerequisite for commitment to terminal differentiation, TGF-beta s inhibited normal keratinization in suprabasal cells and promoted the type of differentiation commonly associated with wound-healing and epidermal hyperproliferation. The actions of TGF-beta s and RA on normal keratinization were synergistic, whereas those on abnormal differentiation associated with hyperproliferation were antagonistic. These observations underscore the notion that environmental changes can act separately on proliferating and differentiating cells within the population. Under the conditions used here, the action of TGF-beta s on human keratinocytes was dominant over RA, and TGF-beta s did not seem to be induced as a consequence of RA treatment. This finding is consistent with the fact that RA accelerated, rather than inhibited, proliferation in raft cultures. Collectively, our data suggest that the effects of both factors on epidermal growth and differentiation are multifaceted and the extent to which their action is coupled in keratinocytes may vary under different conditions and/or in different species.     

Immunofluorescent localization of histone H10 in the nuclei of proliferating and differentiating Friend cells

By using affinity-purified antibodies to H10 and to H1AB the localization of these histones was studied by indirect immunofluorescence in the nuclei of proliferating (EAT and uninduced Friend cells) and of differentiating (induced Friend cells) cell populations. While with H1AB antibodies a bright fluorescence all over the chromatin was obtained, the localization of H10 varied depending on the state of the cell population. In the proliferating EAT cells it was localized strictly in the nucleoli. The Friend cell population revealed a heterogeneous picture with two types of H10 localization-nucleolar predominating in uninduced cell populations and peripheral predominating in induced cells. A comparison with literature data suggests that H10 seems to be associated with chromatin regions containing active genes.     

Detection and Discrimination of Tea Plant Stresses Based on Hyperspectral Imaging Technique at a Canopy Level

Tea plant stresses threaten the quality of tea seriously. The technology corresponding to the fast detection and differentiation of stresses is of great significance for plant protection in tea plantation. In recent years, hyperspectral imaging technology has shown great potential in detecting and differentiating plant diseases, pests and some other stresses at the leaf level. However, the lack of studies at canopy level hampers the detection of tea plant stresses at a larger scale. In this study, based on the canopy-level hyperspectral imaging data, the methods for identifying and differentiating the three commonly occurred tea stresses (i.e., the tea leafhopper, anthrax and sun burn) were studied. To account for the complexity of the canopy scenario, a stepwise detecting strategy was proposed that includes the process of background removal, identification of damaged areas and discrimination of stresses. Firstly, combining the successive projection algorithm (SPA) spectral analysis and K-means cluster analysis, the background and overexposed non-plant regions were removed from the image. Then, a rigorous sensitivity analysis and optimization were performed on various forms of spectral features, which yielded optimal features for detecting damaged areas (i.e., YSV, Area, GI, CARI and NBNDVI) and optimal features for stresses discrimination (i.e., MCARI, CI, LCI, RARS, TCI and VOG). Based on this information, the models for identifying damaged areas and those models for discriminating different stresses were established using K-nearest neighbor (KNN), Random Forest (RF) and Fisher discriminant analysis. The identification model achieved an accuracy over 95%, and the discrimination model achieved an accuracy over 93% for all stresses. The results suggested the feasibility of stress detection and differentiation using canopy-level hyperspectral imaging techniques, and indicated the potential for its extension over large areas.     

Primary culture of medaka (<Emphasis Type="Italic">Oryzias latipes</Emphasis>) testis: a test system for the analysis of cell proliferation and differentiation

An in vitro test system using primary testis cells of the medaka (Oryzias latipes) was established that provides quantitative data on cell proliferation and spermatocyte differentiation. The primary cultures were characterised over a period of 2 days with respect to cell viability and the distribution of adherent and suspended cells. These two cell populations were maintained at a dynamic equilibrium in vitro for several days. The proliferating cells were predominantly present amongst the clusters of suspended cells as determined by BrdU labelling (cytological identification and quantification by ELISA). Based on cytological criteria the proliferating cells were mostly spermatogonia and preleptotene spermatocytes. Differentiation of spermatocytes to spermatids or spermatozoa was also observed mainly amongst suspended cells. Quantification of cell proliferation and cell differentiation by flow cytometry was achieved by labelling the primary cells with carboxyfluorescein diacetate N-succinimidyl ester, which allowed the identification and quantification of meiotically or mitotically dividing primary cells. Addition of the flavonoid genistein (10 µg/ml) to the primary cultures inhibited both cell proliferation and cell differentiation significantly. The test system is suitable for the study of the effect of substances which interfere with spermatogenesis in the vertebrate medaka model.     

Label‐free quantitative chemical imaging and classification analysis of adipogenesis using mouse embryonic stem cells

Stem cells have received much attention recently for their potential utility in regenerative medicine. The identification of their differentiated progeny often requires complex staining procedures, and is challenging for intermediary stages which are a priori unknown. In this work, the ability of label‐free quantitative coherent anti‐Stokes Raman scattering (CARS) micro‐spectroscopy to identify populations of intermediate cell states during the differentiation of murine embryonic stem cells into adipocytes is assessed. Cells were imaged at different days of differentiation by hyperspectral CARS, and images were analysed with an unsupervised factorization algorithm providing Raman‐like spectra and spatially resolved maps of chemical components. Chemical decomposition combined with a statistical analysis of their spatial distributions provided a set of parameters that were used for classification analysis. The first 2 principal components of these parameters indicated 3 main groups, attributed to undifferentiated cells, cells differentiated into committed white pre‐adipocytes, and differentiating cells exhibiting a distinct protein globular structure with adjacent lipid droplets. An unsupervised classification methodology was developed, separating undifferentiated cell from cells in other stages, using a novel method to estimate the optimal number of clusters. The proposed unsupervised classification pipeline of hyperspectral CARS data offers a promising new tool for automated cell sorting in lineage analysis.      

IL-5 induces a Pgp-1 (CD44) bright B cell subpopulation that is highly enriched in proliferative and Ig secretory activity and binds to hyaluronate

Pgp-1 expression was examined in unstimulated B cell populations and in B cells activated with several polyclonal stimuli. Flow cytometry analysis demonstrated that Pgp-1 expression increased when B cells were activated with supernatant of cloned Th2 cells, with LPS, or with IL-5, stimuli that induced polyclonal proliferation and differentiation. IL-5-primed B cells were phenotypically unique and could be divided into two distinct subpopulations based on the brightness of Pgp-1 expression. Furthermore, sterile sorting experiments showed that proliferating and differentiating B cells were highly enriched in a Pgp-1-bright, Ia-dull, B220-dull subpopulation. The possibility that Pgp-1 expressed on activated B cells functions as an adhesion molecule was evaluated by assessing adhesion of activated B cells to defined substrates. It was found that IL-5-activated B cells bound strongly to hyaluronate-coated surface, and this binding was specifically inhibited by anti-Pgp-1 Ab. These findings suggest that Pgp-1 expression is a useful marker which, under defined conditions, identifies the proliferating and differentiating subset of activated B cells. Moreover, the Pgp-1 bright subset of IL-5-primed B cells binds to hyaluronate in a Pgp-1-dependent manner that suggests a potential role of Pgp-1 in the in vivo adherence and trafficking of activated B cells.     

Homeotic factor ATBF1 induces the cell cycle arrest associated with neuronal differentiation

The present study aimed to elucidate the function of AT motif-binding factor 1 (ATBF1) during neurogenesis in the developing brain and in primary cultures of neuroepithelial cells and cell lines (Neuro 2A and P19 cells). Here, we show that ATBF1 is expressed in the differentiating field in association with the neuronal differentiation markers beta-tubulin and MAP2 in the day E14.5 embryo rat brain, suggesting that it promotes neuronal differentiation. In support of this, we show that ATBF1 suppresses nestin expression, a neural stem cell marker, and activates the promoter of Neurod1 gene, a marker for neuronal differentiation. Furthermore, we show that in Neuro 2A cells, overexpressed ATBF1 localizes predominantly in the nucleus and causes cell cycle arrest. In P19 cells, which formed embryonic bodies in the floating condition, ATBF1 is mainly cytoplasmic and has no effect on the cell cycle. However, the cell cycle was arrested when ATBF1 became nuclear after transfer of P19 cells onto adhesive surfaces or in isolated single cells. The nuclear localization of ATBF1 was suppressed by treatment with caffeine, an inhibitor of PI(3)K-related kinase activity of ataxa-telangiectasia mutated (ATM) gene product. The cytoplasmic localization of ATBF1 in floating/nonadherent cells is due to CRM1-dependent nuclear export of ATBF1. Moreover, in the embryonic brain ATBF1 was expressed in the cytoplasm of proliferating stem cells on the ventricular zone, where cells are present at high density and interact through cell-to-cell contact. Conversely, in the differentiating field, where cell density is low and extracellular matrix is dense, the cell-to-matrix interaction triggered nuclear localization of ATBF1, resulting in the cell cycle arrest. We propose that ATBF1 plays an important role in the nucleus by organizing the neuronal differentiation associated with the cell cycle arrest.     

Subtype-specific neuronal differentiation of PC12 cells transfected with alpha2-adrenergic receptors

Cells of the PC12 rat pheochromocytoma cell line acquire characteristics of sympathetic neurons under appropriate treatment. Stably transfected PC12 cells expressing individual alpha2-adrenergic receptor (alpha2-AR) subtypes were used to assess the role of alpha2-ARs in neuronal differentiation and to characterise the signalling pathways activated by the alpha2-AR agonist epinephrine in these cells. The effects of alpha2-AR activation were compared with the differentiating action and the signalling mechanisms of nerve growth factor (NGF). Epinephrine induced neuronal differentiation of PC12alpha2 cells through alpha2-AR activation in a subtype-dependent manner, internalization of all human alpha2-AR subtypes, and activation of mitogen-activated protein kinase (MAPK) and the serine-threonine protein kinase Akt. Epinephrine and NGF showed synergism in their differentiating effects. The MAPK kinase (MEK-1) inhibitor PD 98059 abolished the differentiating effect of epinephrine indicating that the differentiation is dependent on MAPK activation. Activating protein-1 (AP-1) DNA-binding activity was increased after epinephrine treatment in all three PC12alpha2 subtype clones. Evaluation of the potential physiological consequences of these findings requires further studies on endogenously expressed alpha2-ARs in neuronal cells.     

Adult human brain neural progenitor cells (NPCs) and fibroblast-like cells have similar properties in vitro but only NPCs differentiate into neurons

The ability to culture neural progenitor cells from the adult human brain has provided an exciting opportunity to develop and test potential therapies on adult human brain cells. To achieve a reliable and reproducible adult human neural progenitor cell (AhNPC) culture system for this purpose, this study fully characterized the cellular composition of the AhNPC cultures, as well as the possible changes to this in vitro system over prolonged culture periods. We isolated cells from the neurogenic subventricular zone/hippocampus (SVZ/HP) of the adult human brain and found a heterogeneous culture population comprised of several types of post-mitotic brain cells (neurons, astrocytes, and microglia), and more importantly, two distinct mitotic cell populations; the AhNPCs, and the fibroblast-like cells (FbCs). These two populations can easily be mistaken for a single population of AhNPCs, as they both proliferate under AhNPC culture conditions, form spheres and express neural progenitor cell and early neuronal markers, all of which are characteristics of AhNPCs in vitro. However, despite these similarities under proliferating conditions, under neuronal differentiation conditions, only the AhNPCs differentiated into functional neurons and glia. Furthermore, AhNPCs showed limited proliferative capacity that resulted in their depletion from culture by 5-6 passages, while the FbCs, which appear to be from a neurovascular origin, displayed a greater proliferative capacity and dominated the long-term cultures. This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures. These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations. This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting AhNPC-based experiments.     

Cell proliferation and differentiation kinetics during spermatogenesis inHydra carnea

Summary Spermatogenesis inHydra carnea was investigated. The cell proliferation and differentiation kinetics of intermediates in the spermatogenesis pathway were determined, using quantitative determinations of cell abundance, pulse and continuous labelling with³H-thymidine and nuclear DNA measurements. Testes develop in the ectoderm of male hydra as a result of interstitial cell proliferation. Gonial stem cells and proliferating spermatogonia have cell cycles of 28 h and 22 h, respectively. Stem cells undergo four, five or six cell divisions prior to meiosis which includes a premeiotic S+G2 phase of 20 h followed by a long meiotic prophase (22 h).Spermatid differentiation requires 12–29 h. When they first appear, testes contain only proliferating spermatogonia; meiotic and postmeiotic cells appear after 2 and 3 days, respectively and release of mature sperm begins after 4 days. Mature testes produce about 27,000 sperm per day over a period of 4–6 days: about 220 gonial stem cells per testis are required to support this level of sperm differentiation. Further results indicate that somatic (e.g. nematocyte) differentiation does not occur in testes although it continues normally in ectodermal tissue outside testes. Our results support the hypothesis that spermatogenesis is controlled locally in regions of the ectoderm where testes develop.     

A mathematical model for assessing changes in neurofilament protein levels in neurites and cell bodies of differentiating neuroblastoma cells

A mathematical model which allows the calculation of the level of neurofilament protein in the cell body (x) and in the neurites (y) of differentiating SK-N-SH cells is presented. The model considers the changes in cell number (proliferating cells) and the number of cells with neurites (differentiating cells). It takes into account the fact that (i) when cells are cultured in differentiating conditions, an increase in cell number is initially observed and (ii) in a non-synchronized population of differentiating cells, the length of neurite extended by individual cells varies within the population. Total neurofilament protein levels in a population of cells were measured by enzyme-linked immunoabsorbant assay and application of the model to the data allowed values for x and y to be calculated. The validity of the model is supported by the fact that the predicted total neurofilament protein levels are highly correlated with the experimentally derived neurofilament protein levels. The model should be of use in temporal studies of cytoskeletal proteins involved in neuronal growth/differentiation and also in studies in which the system is a target of toxic insult.     

Expression of the nuclear protein mitotin in differentiating in vitro HL 60 cells.

Mitotin is a 125 kDa/pI 6.5 nuclear protein specific for proliferating cells and markedly increased prior to and during mitosis. This study presents evidence for the expression of this protein during dimethylsulfoxide (DMSO) induced differentiation of human promyelocytic leukemia HL 60 cells. The expression had been followed at two levels: as antigen, using a specific antimitotin monoclonal antibody and as mRNA, using a specific cDNA probe. The results from the immunofluorescent study show a gradual disappearance of mitotin in differentiating HL 60 cells starting from the fourth day after DMSO induction. On the other hand, the changes in the expression of mitotin mRNA were much more dramatic. This mRNA is expressed at a high level during the first three days of differentiation but shows a striking decrease after the fourth day. This correlates with the rapid changes in the number of blast cells in the differentiating HL 60 cell population. Therefore, the expression of mitotin mRNA can serve as a marker for the changes accompanying the termination of cell proliferation in differentiating cells.     

Characterisation of myogenic cell membrane: II. Dynamic changes in membrane lipids during the differentiation of mouse C2 myoblast cells

Proliferating mouse C2 myoblast cells resist haemagglutinating virus of Japan, Sendai virus (HVJ) mediated cell fusion. However, differentiating C2 cells can be induced to fuse by HVJ, suggesting that the rigid membrane of C2 cells changes during the differentiation. To investigate this phenomenon, changes in membrane lipids which affect fluidity were examined. Membrane cholesterol gradually decreased with the differentiation of C2 cells. However, spontaneous fusion to form myotubes and artificial fusion induced by HVJ were both inhibited when the level of cholesterol was prevented from falling in the cell membrane. The membranes of differentiating C2 cells contained more unsaturated fatty acids than those of proliferating cells. Thus, when differentiating C2 cells were treated with stearate (a saturated fatty acid), they failed to form myotubes and were insensitive to HVJ-mediated fusion. Whereas, if proliferating C2 cells were given linolenate (an unsaturated fatty acid), they became capable of HVJ-induced fusion. These results indicate that differentiating C2 cells change their fusion sensitivity by decreasing cholesterol, probably at the same time as they increase the unsaturated fatty acid content of the cell membrane.     

In Vitro Modeling of the Neurovascular Environment by Coculturing Adult Human Brain Endothelial Cells with Human Neural Stem Cells

Brain and vascular cells form a functionally integrated signalling network that is known as the neurovascular unit (NVU). The signalling (autocrine, paracrine and juxtacrine) between different elements of this unit, especially in humans, is difficult to disentangle in vivo. Developing representative in vitro models is therefore essential to better understand the cellular interactions that govern the neurovascular environment. We here describe a novel approach to assay these cellular interactions by combining a human adult cerebral microvascular endothelial cell line (hCMEC/D3) with a fetal ganglionic eminence-derived neural stem cell (hNSC) line. These cell lines provide abundant homogeneous populations of cells to produce a consistently reproducible in vitro model of endothelial morphogenesis and the ensuing NVU. Vasculature-like structures (VLS) interspersed with patches of differentiating neural cells only occurred when hNSCs were seeded onto a differentiated endothelium. These VLS emerged within 3 days of coculture and by day 6 were stabilizing. After 7 days of coculture, neuronal differentiation of hNSCs was increased 3-fold, but had no significant effect on astrocyte or oligodendrocyte differentiation. ZO1, a marker of adherens and tight junctions, was highly expressed in both undifferentiated and differentiated endothelial cells, but the adherens junction markers CD31 and VE-cadherin were significantly reduced in coculture by approximately 20%. A basement membrane, consisting of laminin, vitronectin, and collagen I and IV, separated the VLS from neural patches. This simple assay can assist in elucidating the cellular and molecular signaling involved in the formation of VLS, as well as the enhancement of neuronal differentiation through endothelial signaling.