In vitro differentiation of human retinoblastoma cells into neuronal phenotypes

In order to characterize the cell type(s) of origin of human retinoblastoma cells by immunophenotyping, primary cells from seven retinoblastomas and of the corresponding cell lines (RBL lines), as well as four retinoblastoma (RB) lines established by other groups, were compared with rat and human retina cells, and with the adenovirus E1A-transformed human retinoblast cell line HER-Xho1-CC2. Analyses using monoclonal antibodies (Mabs) RB13-2 and RB21-7, originally raised against prenatal rat brain cells and recognizing neural cell surface antigens expressed in a developmental-stage-dependent manner, and three cell-type-specific Mabs (Q211, M501, Mab directed against vimentin) developed by other groups, gave the following results: (i) Retinoblastomas consist of cells expressing differentiated neuronal phenotypes during cultivation in vitro; (ii) All of the newly established RBL lines express neuronal phenotypes; and (iii) Cell lines such as Y79, which have been propagated in vitro for extended periods, do not express antigens specific for the neuronal pathway and cannot, therefore, be considered phenotypically representative of retinoblastoma cells.     

The expression and potential function of cellular prion protein in human lymphocytes

We examined expression of the normal cellular prion protein (PrP(C)) in human peripheral blood mononuclear cells (PBMC) and in transfected neuroblastoma cells with a panel of six monoclonal antibodies (Mabs). While all six of the Mabs reacted strongly with the neuroblastoma cells, only four of the Mabs reacted with PrP(C) expressed by human PBMC. PrP(C) is expressed at high levels in human T cells, B cells, monocytes, and dendritic cells, but not in red blood cells. Immunoblotting studies revealed that the PrP(C) glycoforms and the composition of the N-linked glycans on PrP(C) in human PBMC are different from those of the brain or the neuroblastoma cells. In human PBMC and the neuroblastoma cell lines the N-terminal portion of the PrP(C) is hypersensitive to proteolytic digestion, suggesting that the N-terminus of the PrP(C) on the surface of a living cell lacks secondary structure. We found that the level of PrP(C) expressed on the surface of human T lymphocytes was up-regulated as a consequence of cellular activation. Accordingly, memory T cells express more PrP(C) than na?ve T cells. In addition, the proliferation of human T lymphocytes stimulated with an anti-CD3 Mab was inhibited by anti-PrP(C) Mabs. Collectively, these results suggest that PrP(C) can participate in signal transduction in human T lymphocytes.     

Generation and regulation of developing immortalized neural cell lines

The genetic and environmental signals that regulate progressive lineage elaboration in the mammalian brain are poorly understood. In addition, characterization of the developmental profiles of early central nervous system (CNS) stem/ progenitor cells and analysis of the mechanisms involved in their clonal expansion, lineage restriction, and cellular maturation have been fragmentary and elusive. These seminal neurodevelopmental issues have been examined using a series of clonally derived neural stem/progenitor cell lines established by retroviral transduction of embryonic (E16.5-E17.5) murine hippocampal and cerebellar cells using temperature-sensitive alleles (A58/U19) of the simian virus (SV) 40 large tumor (T) antigen. Under conditions permissive for T-antigen expression (33 degrees C), single neural stem cells exhibited self-renewal, clonal expansion, and both symmetric and asymmetric modes of cell division. By contrast, at the nonpermissive temperature for T-antigen expression (39 degrees C), specific sets of cytokines potentiated the progressive elaboration of neuronal, oligodendroglial, and astroglial lineage species. These observations demonstrate that a spectrum of genetic and epigenetic signals and distinct cellular processes are involved in orchestrating the evolution of individual neural lineages from regional CNS stem/progenitor species. Further, the availability of conditionally immortalized neural cell lines that can be transplanted back into the mammalian brain may represent an important experimental resource for the detailed characterization of cellular and molecular mechanisms involved in the developmental sculpting, plasticity, and regeneration of the mammalian CNS.     

Expression of CD13/aminopeptidase N in precursor B-cell leukemia: role in growth regulation of B cells

Expression of cell surface CD13 in acute B-cell leukemia (ALL-B) is often viewed, as an aberrant expression of a myeloid lineage marker. Here, we attempted to study the stage specific expression of CD13 on ALL-B blasts and understand its role in leukemogenesis as pertaining to stage of B-cell ontogeny. A total of 355 cases of different hematological malignancies were diagnosed by immunophenotyping. Among 68 cases of early B-cell ALL, 22 cases with distinct immunophenotype was identified as immature B-cell ALL. Blasts from these ALL-B patients demonstrated prominent expression of CD10, CD19, CD22, but neither cytoplasmic nor surface IgM receptors. This strongly indicates leukemogenesis at an early stage of B-cell development. We also identified, the existence of a subpopulation of cells with remarkably similar phenotype in non-leukemic marrow from healthy subjects (expressing CD10, CD19, CD22, CD24, Tdt together with the co-expression of CD13). This sub-population of B cells concomitantly expressing CD13 appeared to be a highly proliferating group. By blocking their cell surface CD13 in leukemic blasts with monoclonal antibody we were able to inhibit their proliferation. We hypothesized that neoplastic transformation at this stage may be facilitated by CD13. CD13 may thus be an important target for novel molecular therapy of early stage acute B-cell leukemia.     

Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin

The derivation and characterization of 22 hybridoma clones producing monoclonal antibodies (Mabs) specific for the transforming protein of Rous sarcoma virus, pp60src, are described. All Mabs reacted with pp60v-src encoded by Prague, Schmidt-Ruppin, and Bratislava 77 strains of Rous sarcoma virus. Of these Mabs, 10 efficiently immunoprecipitated pp60c-src from chicken embryo cells. Of these 10 Mabs, 2 (GD11 and EB8) readily detected pp60c-src from a variety of rodent and human cultured cells and from rat brain tissue in an in vitro immune complex kinase assay. Mapping experiments have tentatively localized the determinant(s) recognized by GD11 and EB8 to a region of the src protein bounded by amino acid residues 82 to 169, whereas the remaining Mabs appeared to recognize determinants residing within residues 1 to 82 or 169 to 173. Most of the Mabs complexed denatured pp60v-src in a Western immunoblot, and several were used to localize pp60v-src in Rous sarcoma virus-transformed chicken embryo cells by indirect immunofluorescence microscopy.     

Stage-specific changes in gene expression in acutely isolated mouse CNS progenitor cells

Neural progenitor cells can be derived from a variety of developmental stages when they are preferentially proliferating, undergoing neurogenesis or undergoing gliogenesis. We used FACS sorting and the LeX surface marker to enrich neural progenitor cells from different embryonic stages and adult and compared their gene expression profiles using Affymetrix Microarrays. Our results show that, while there are common genes expressed in the progenitor cell population from all stages, there are also significant differences in gene expression patterns that correlate with stage-related behaviors. These data indicate that progenitor cells change during development and that adult and embryonic neural progenitor cells are intrinsically different.     

EFFECT OF ACTINOMYCIN D ON THE DIFFERENTIATION OF HATCHING GLAND CELL AND CILIA CELL IN THE FROG EMBRYO

The forehead epidermis of the stage 18–20 R. japonica embryo includes the hatching gland cell (HGC) which contains cell-specific secretory granules. The cilia cell (CC) and common epidermal cell (CEC) constitute the epidermis of the entire body surface, in addition to the forehead region.
Culture of superficial epidermal explants from various embryonic portions at various developmental stages revealed that HGCs are derived from cells localized on the neural crest in the stage 13a (early neural plate) embryo. When explants from the presumptive HGC area were treated with 1 ug/ml actinomycin D (AMD), the formation of secretory granules in HGCs was inhibited either by continuous treatment from stage 13 or by an 8-hr treatment at stage 13b. Similarly, the ciliogenesis in CCs was inhibited. The differentiation of CECs was entirely unaffected by any of the AMD treatment. After release from AMD, mucous vesicles, characteristic of the CEC, were formed in cells whose differentiation into HGC and CC had been suppressed by the antibiotic. Thread complexes and clumps of coiled strings were found in the nuclei of AMD-affected cells.
It is concluded that the DNA-dependent RNA syntheses which direct secretory granule formation in the HGC and ciliogenesis in the CC occur during a limited period at stage 13b, viz. , 20 hr before their cytodifferentiation becomes appreciable.     

Expression of neural cell adhesion molecules,NCAMs, and their polysialylated forms,PSA-NCAMs,in the developing rat pituitary gland

Neural cell adhesion molecules (NCAMs) can undergo post-translational modifications, such as the addition of polysialic acid chains, thus generating PSANCAMs, which are expressed mainly during development. Since polysialylation considerably modifies NCAM adhesivity, expression of NCAMs and PSANCAMs has been investigated in the developing hypophysis by immunohistochemistry. At embryonic day 13 (E13), an antibody against NCAM outlined all cellular profiles in the entire Rathke's pouch; this labelling persisted until adulthood. NCAM expression increased in all lobes during development and concerned all pituitary cell types. In contrast, at E13, PSA-NCAMs were only detected in the neural lobe, solely constituted of pituicytes at this stage, and the tuberal lobe, the only lobe expressing hormonal mRNA at the same stage. PSA-NCAMs expression increased in the neural lobe at E17 with the arrival of the neurosecretory fibres and persisted into adulthood. In the anterior lobe, PSA-NCAMs appeared at E15 where their distribution was similar to that of the differentiating corticotrophic cells; at subsequent stages, their expression extended to the whole anterior lobe. Only two cell types, corticotrophic and somatotrophic cells, remained labelled in the adult gland. In the intermediate lobe, melanotrophic cells never expressed PSA-NCAMs but these were expressed on folliculo-stellate cells at birth, preceding the onset of innervation. These results suggest that NCAMs and PSA-NCAMs play a role in pituitary histogenesis, cell differentiation and neurointermediate lobe innervation.     

Transcriptional Profiling of Adult Neural Stem-Like Cells from the Human Brain

There is a great potential for the development of new cell replacement strategies based on adult human neural stem-like cells. However, little is known about the hierarchy of cells and the unique molecular properties of stem- and progenitor cells of the nervous system. Stem cells from the adult human brain can be propagated and expanded in vitro as free floating neurospheres that are capable of self-renewal and differentiation into all three cell types of the central nervous system. Here we report the first global gene expression study of adult human neural stem-like cells originating from five human subventricular zone biopsies (mean age 42, range 33–60). Compared to adult human brain tissue, we identified 1,189 genes that were significantly up- and down-regulated in adult human neural stem-like cells (1% false discovery rate). We found that adult human neural stem-like cells express stem cell markers and have reduced levels of markers that are typical of the mature cells in the nervous system. We report that the genes being highly expressed in adult human neural stem-like cells are associated with developmental processes and the extracellular region of the cell. The calcium signaling pathway and neuroactive ligand-receptor interactions are enriched among the most differentially regulated genes between adult human neural stem-like cells and adult human brain tissue. We confirmed the expression of 10 of the most up-regulated genes in adult human neural stem-like cells in an additional sample set that included adult human neural stem-like cells (n = 6), foetal human neural stem cells (n = 1) and human brain tissues (n = 12). The NGFR, SLITRK6 and KCNS3 receptors were further investigated by immunofluorescence and shown to be heterogeneously expressed in spheres. These receptors could potentially serve as new markers for the identification and characterisation of neural stem- and progenitor cells or as targets for manipulation of cellular fate.     

Elements of the nitric oxide/cGMP pathway expressed in cerebellar granule cells: biochemical and functional characterisation

It is known that the nitric oxide (NO)/cGMP pathway affects neuronal development and the expression of the different proteins is developmentally dependent in several brain areas. However, so far there are no data on the expression of the proteins involved in this signalling system during the development of the cerebellar granule cell, one of the most widely used models of neuronal development. This study was accordingly designed to analyse the developmental regulation of neuronal nitric oxide synthase (nNOS), soluble guanylyl cyclase subunits (alpha1, alpha2 and beta1) and cGMP-dependent protein kinases (cGK I and cGK II) in cerebellar granule cells through real time-polymerase chain reaction (RT-PCR) and Western blotting. We were able to detect guanylyl cyclase subunits and cGK I and cGK II in cerebellar granule cells at every stage of development examined (cells freshly isolated from 7-day-old rat pups, and cells cultured for 7 days or 14 days). Expression levels, nevertheless, varied significantly at each stage. nNOS, alpha2 and beta1 and cGK II levels increased during granule cell development, while alpha1 and cGK I showed an opposite behaviour pattern; the levels of these latter proteins diminished as the cells matured. The functionality of this pathway was assessed by stimulating cells kept in culture for 7 days with DEA/NO or with N-methyl-D-aspartate (NMDA). Cells responded by increasing intracellular cGMP and activating cGMP-dependent protein kinase activity, which effectively phosphorylated two well-known substrates of this activity, the vasodilator stimulated phosphoprotein (VASP) and the cAMP response element binding protein (CREB). In summary, through both functional and biochemical tests, this is the first demonstration of a complete NO/cGMP signalling transduction pathway in cerebellar granule cells. Our results also indicate the developmental regulation of the proteins in this system.     

Maternal zinc deficiency impairs brain nestin expression in prenatal and postnatal mice

INTRODUCTIONZinc is essential for normal brain development,evidenced by the fact that zinc deficiency in lactating mothers is characterized by a high incidence ofneuroanatomical maiformatinns and functional abnormalities in suckling offspring[1-3]. By colltrast,relatively little is known about the relationship be{tween maternal zinc nutrition and fetal brain development[2, 4, 5]. Dvergsten et al[6-81 investigated theeffects of maternal zinc deficiency on postnatal development of the rat ce…     

Expression of idiotype 315 (Id315) and I-A antigens in MOPC-315 tumor cells grown in vivo

MOPC-315 murine myeloma grown intraperitoneally as ascites cells in BALB/cJ mice were removed at successive days after transplantation. They were stained for Id³¹⁵ and I-A markers by indirect immunofluorescence techniques by means of FITC conjugates. Flow cytometry (FCM) measurements on cell surface markers were correlated with the phase of the cell cycle by quantitating cellular DNA of cells stained with propidium iodide. Variations in cell size due to cell growth were determined by low-angle light scattering. FCM data on the two cell surface markers were normalized for unit cell surface and cell volume. Cells grew rapidly in the early days (5–7) of tumor growth. No significant variation in the expression of surface markers was observed during this period. Parallel with a slowdown in cell growth, the expression of Id³¹⁵ increased about threefold between the 7th and 9th days. The increase in the Id³¹⁵ marker was dependent on the cell cycle, with G₁ cells having the highest density. No cell cycle dependence was observed for the I-A marker.     

Relations between pigmentation of the cornea and the number of epidermal melanophores in Rana temporaria L. larvae

The dynamics of the external cornea pigmentation in Rana temporaria L. larvae at the 22d developmental stage have been studied under conditions favourable for various course of certain morphological reactions in the pigment system. The cornea together with the surrounding skin is transferred on the dorsal surface of the larva body, and the piece of the dorsal surface skin is put instead of the cornea removed. When using the reciprocal transplantation method and preserving the organism's integrity (without disturbing melanocyte-stimulating source--namely, the hypophysis, and melatonine sources--namely, the pineal gland and the lateral eyes) the corneal pigmentation is observed on the background of perfect morphological reactions in the pigment system, while the larvae are maintained on the dark and light substrates, that is at various density of the pigment cells (120 larvae have been used). The pigmentation dynamics have been studied from the 6th up to the 20th day in total preparations. The epidermal melanophores density is estimated in 4 areas of each preparation. The melanin amount is estimated by means of the electron paramagnetic resonance-spectrometry according to the contents of free radicals expressed in relative units. A direct proportional dependence between the significantly higher melanin contents (1.5-fold) and a significantly quicker (1.5-fold) process of the corneal pigmentation is revealed, that agrees with an increasing number of the pigment cells per one unit of the body surface in the larvae maintained on the dark substrate. In the larvae maintained on the light substrate, the dependence is of a reverse character. It is probable that the factors forcing the pigmented cells, at cultivation the neural crest cells in vitro to reject from each other, affect the pigmentation of the larval cornea in vivo. If it is the case, the processes specific for the embryonal period, transgress during the cornea pigmentation at the larval stages of development.     

Appearance of B and H blood group antigens in the developing cochlear hair cells

Summary The presence of human blood-group antigens was analyzed in the rat cochlea during its postnatal development, using anti-A, anti-B and anti-H antibodies. At no stage was reactivity with anti-A antibody observed. With the anti-H antibody, a strong reactivity was observed from 1 to 9 days after birth within hair cells and some other surface epithelial cells of the cochlear duct. After postnatal day 9, only a faint reactivity persisted in a few non-sensory cells. With the anti-B antibody, only hair cells were selectively labeled. At early stages (postnatal day 1 and 3), the reactivity was intense and observed both around the cell surface and within the supranuclear region of cytoplasm. Later on, the reactivity decreased; it was limited at postnatal day 9 to a reactive spot below the cuticular plate. Results are compared with a preliminary finding describing the first appearance of B and H antigens in the organ of Corti at a prenatal stage, and with data concerning other sensory and neural structures. The appearance and progressive disappearance of B and H antigens on sensory and non-sensory cells can be correlated with significant events in the development of the cochlea. The transient expression of B and H antigens in cochlear sensory cells may correspond to developmental changes in their surface glycoconjugates.     

Development of the tongue and taste disks in Pelobates fuscus

In the tadpole of Pelobates fuscus the process of tongue formation starts at the 32nd developmental stage. In more advanced stages (older than 38th) fast anterior and faucial growth of the tongue fold has been observed. This process is accompanied by the development of the gustatory organs. The dorsal surface of the tongue fold, smooth at the beginning, in older tadpoles (developmental stages 36-39th) forms protrusions in which gustatory organs of the taste disk type (TDs) develop. In the 41 st tadpole developmental stage anlages of TDs are formed by elongated cells, located more or less perpendicularly to the surface of the tongue. The diameter of the sensory area of a TD at the 45th developmental stage amounts to 94 microm, while in metamorphosed individuals it reaches 130-140 microm. At the base of a TD the presence of basal cell morphologically similar to that of Merkel cell was observed at the 42nd developmental stage of a tadpole. Fully developed afferent synaptic connections in the sensory epithelium of a TD were found starting from the 44th developmental stage. Single synaptic vesicles with an electron-dense core were observed in gustatory cells as early as at the 41 st developmental stage of the tadpole. From the observations reported here it can be inferred that in Pelobates fuscus development of both the tongue and TDs is similar to that already described in the representatives of the Rana genus.     

Neural progenitor genes. Germinal zone expression and analysis of genetic overlap in stem cell populations

The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.     

Ontogenetic analysis of some surface markers on pig lymphocytes using fluorescence-activated cell sorter

Surface markers were demonstrated on pig lymphocytes using anti-T cell-IgG and anti-Helix pomatia (HP) IgG during prenatal and postnatal development. A fluorescence-activated cell sorter analysis of T-cell surface markers was accompanied by an image analysis to prove the association of T antigenic determinants with the plasma membrane only. We found development-dependent changes in both anti-T cell and HP surface markers in both primary and secondary lymphatic organs. The number of T-positive (T+) cells estimated by anti-T cell-IgG was very similar to the results obtained by spontaneous E-rosette forming tests. At all selected age intervals, changes in the number of T+ cells were not significant in the thymus, but a marked increase in T+ cells was found in both spleen and lymph nodes. The image analysis confirmed the expression of T cell markers on the cell surface. The distribution of T cell markers was uneven, i.e. various degree of fluorescence intensity on whole ring-pattern projection of the cell surface image was estimated. In second lymphatic organs especially, fluorescence intensity of cells, i.e. total number of T cell markers estimated by anti-T cell-IgG, increased with age. On fetal day 73, T cell markers were slightly expressed, but very high fluorescence intensity and heterogeneous distribution of T cell markers on lymphocytes were found on fetal day 107 and postnatal day 56. The results indicate the possibility of functional maturation of various T cell markers on T cell subsets, furthermore a different degree of expression of T cell markers on various T cell subsets can be suggested. The number of HP+ cells increased with age in both primary and secondary lymphatic organs. In the prenatal period, the expression of HP receptors was very weak in both primary and secondary organs in contrast to the marked increase in HP+ cells during the postnatal interval. Differences in fluorescence intensity of cells were found, representing the increase by 22% in thymus cells comparing to cells of secondary lymphatic organs. Heterogeneity of HP+ cell populations in thymus was shown by the Scatchard plot, indicating at least two subpopulations of HP+ cells with different avidity to HP. Cells with low HP avidity could include a subset with cytolytic activity.     

Somatic variation of H-2Kk expression and structure in a T-cell lymphoma: instability, stabilization, high production and structural mutation

In the heterozygous T lymphoma line LDHB, variants which have lost the expression of individual H-2 class I genes are spontaneously generated in vitro at a frequency of 10(-1)-10(-2). A cell line (HK13) in which the class I gene Kk is stably expressed (frequency of loss variants less than 10(-4) was selected from LDHB cells by fluorescence activated cell sorting. Further selection of HK13 cells for high Kk expression led to the isolation of the HK22 line which expresses twice as much Kk as HK13. From HK13 and HK22 cells, spontaneous structural variants of Kk having lost individual serological determinants of the Kk wild-type molecule, were isolated by fluorescence activated cell sorting. Such variants occur at a frequency of 10(-6)-10(-7) per cell per generation. The analysis of these variants indicates that they carry mutations in the Kk structural gene and that HK13 cells express a single Kk gene which appears to be duplicated in HK22 cells. We did not find evidence for the generation of variants expressing 'alien' class I products in the LDHB cell line. The instability of class I gene expression in LDHB cells and the transition to stable expression may represent steps of T-cell differentiation in the thymus.