Glioblastoma cells inhibit astrocytic p53-expression favoring cancer malignancy.

The tumor microenvironment has a dynamic and usually cancer-promoting function during all tumorigenic steps. Glioblastoma (GBM) is a fatal tumor of the central nervous system, in which a substantial number of non-tumoral infiltrated cells can be found. Astrocytes neighboring these tumor cells have a particular reactive phenotype and can enhance GBM malignancy by inducing aberrant cell proliferation and invasion. The tumor suppressor p53 has a potential non-cell autonomous function by modulating the expression of secreted proteins that influence neighbor cells. In this work, we investigated the role of p53 on the crosstalk between GBM cells and astrocytes. We show that extracellular matrix (ECM) from p53(+/-) astrocytes is richer in laminin and fibronectin, compared with ECM from p53(+/+) astrocytes. In addition, ECM from p53(+/-) astrocytes increases the survival and the expression of mesenchymal markers in GBM cells, which suggests haploinsufficient phenotype of the p53(+/-) microenvironment. Importantly, conditioned medium from GBM cells blocks the expression of p53 in p53(+/+) astrocytes, even when DNA was damaged. These results suggest that GBM cells create a dysfunctional microenvironment based on the impairment of p53 expression that in turns exacerbates tumor endurance.

Cycle arrest and aneuploidy induced by zidovudine in murine embryonic stem cells.

Zidovudine (3'-azido-3'-deoxythymidine; AZT) is a nucleoside analogue widely used for the treatment of acquired immune deficiency syndrome (AIDS). Medical guidelines recommend the use of AZT by pregnant women in order to reduce risk of HIV vertical transmission. Although it is efficacious, little is known about the side effects of AZT on embryonic development. In this sense, we used murine embryonic stem (mES) cells as a model to investigate the consequences of AZT exposure for embryogenesis. Firstly, mES colonies were incubated with AZT (50 or 100 µM) and cell cycle profile was evaluated. While 27.7 ± 5.43% of untreated mES cells were in G2/M phase, this percentage raised to 45.96 ± 4.18% after AZT exposure (100 µM). To identify whether accumulation of cells in G2/M phase could be related to chromosome missegregation with consequent cell cycle arrest, aneuploidy rate was evaluated after AZT treatment. Untreated colonies presented 39.6 ± 8.4% of cells aneuploid, while after AZT 100 µM treatment, the proportion of aneuploid cells raised to 67.8 ± 3.4% with prevalence of chromosome loss. This event was accompanied by micronuclei formation as AZT 100 µM treated mES cells presented a 2-fold increase compared to untreated ones. These data suggest that AZT exerts genotoxic effects and increases chromosome instability at early stages of embryonic development.

Worldwide survey of published procedures to culture human embryonic stem cells.

Since their derivation 11 years ago, human embryonic stem (hES) cells have become a powerful tool in both basic biomedical research and developmental biology. Their capacity for self-renewal and differentiation into any tissue type has also brought interest from fields such as cell therapy and drug screening. We conducted an extensive analysis of 750 papers (51% of the total published about hES cells between 1998 and 2008) to present a spectrum of hES cell research including culture protocols developed worldwide. This review may stimulate discussions about the importance of having unvarying methods to culture hES cells, in order to facilitate comparisons among data obtained by research groups elsewhere, especially concerning preclinical studies. Moreover, the description of the most widely used cell lines, reagents, and procedures adopted internationally will help newcomers on deciding the best strategies for starting their own studies. Finally, the results will contribute with the efforts of stem cell researchers on comparing the performance of different aspects related to hES cell culture methods.

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel™-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system.

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

Aneuploid neurons are functionally active and integrated into brain circuitry.

The existence of aneuploid cells within the mammalian brain has suggested the influence of genetic mosaicism on normal neural circuitry. However, aneuploid cells might instead be glia, nonneural, or dying cells, which are irrelevant to direct neuronal signaling. Combining retrograde labeling with FISH for chromosome-specific loci, distantly labeled aneuploid neurons were observed in expected anatomical projection areas. Coincident labeling for immediate early gene expression indicated that these aneuploid neurons were functionally active. These results demonstrate that functioning neurons with aneuploid genomes form genetically mosaic neural circuitries as part of the normal organization of the mammalian brain.

Genetics and cell biology of lysophosphatidic acid receptor-mediated signaling during cortical neurogenesis.

Lysophosphatidic acid (LPA) is a small lysophospholipid that signals through G-protein coupled receptors (GPCRs) to mediate diverse cellular responses. Two LPA receptors, LPA(1) and LPA(2), show gene expression profiles in mouse embryonic cerebral cortex, suggesting roles for LPA signaling in cerebral cortical development. Here, we review loss-of-function and gain-of-function models that have been used to examine LPA signaling. Genetic deletion of lpa(1) or both lpa(1) and lpa(2) in mice results in 50-65% neonatal lethality, but not obvious cortical phenotypes in survivors, suggesting that compensatory signaling systems exist for regulating cortical development. A gain-of-function model, approached by increasing receptor activation through exogenous delivery of LPA, shows that LPA signaling regulates cerebral cortical growth and anatomy by affecting proliferation, differentiation and cell survival during embryonic development.

Chromosomal variation in neurons of the developing and adult mammalian nervous system.

A basic assumption about the normal nervous system is that its neurons possess identical genomes. Here we present direct evidence for genomic variability, manifested as chromosomal aneuploidy, among developing and mature neurons. Analysis of mouse embryonic cerebral cortical neuroblasts in situ detected lagging chromosomes during mitosis, suggesting the normal generation of aneuploidy in these somatic cells. Spectral karyotype analysis identified approximately 33% of neuroblasts as aneuploid. Most cells lacked one chromosome, whereas others showed hyperploidy, monosomy, and/or trisomy. The prevalence of aneuploidy was reduced by culturing cortical explants in medium containing fibroblast growth factor 2. Interphase fluorescence in situ hybridization on embryonic cortical cells supported the rate of aneuploidy observed by spectral karyotyping and detected aneuploidy in adult neurons. Our results demonstrate that genomes of developing and adult neurons can be different at the level of whole chromosomes.

Differentiation-dependent sensitivity to cell death induced in the developing retina by inhibitors of the ubiquitin-proteasome proteolytic pathway.

The effects of inhibitors of proteasome function were studied in the retina of developing rats. Explants from the retina of neonatal rats at postnatal day (P) 3 or P6 were incubated with various combinations of the proteasome inhibitor carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132), the protein synthesis inhibitor anisomycin, or the adenylyl cyclase activator forskolin. MG132 induced cell death in a subset of cells within the neuroblastic (proliferative) layer of the retinal tissue. The cells sensitive to degeneration induced by either MG132 or anisomycin, were birthdated by bromodeoxyuridine injections. This showed that the MG132-sensitive population includes both proliferating cells most likely in their last round of cell division, and postmitotic undifferentiated cells, at a slightly earlier stage than the population, sensitive to anisomycin-induced cell death. The results show that sensitivity to cell death induced by proteasome inhibitors defines a window of development in the transition from the cell cycle to the differentiated state in retinal cells.

Selective sensitivity of early postmitotic retinal cells to apoptosis induced by inhibition of protein synthesis.

In previous work we showed that apoptosis in retinal tissue from developing rats can be induced by inhibition of protein synthesis (Rehen et al. 1996, Development, 122, 1439-1448). Here we show that recent postmitotic cells are the cells sensitive to apoptosis triggered by blockade of protein synthesis. To label all proliferating cells in the retina, a series of injections of the nucleotide analogue, bromo-deoxy-uridine (BrdU, 60 mg/kg b.w.), was given in rat pups. Then, explants of the retina were incubated in vitro with the inhibitor of protein synthesis anisomycin (1.0-3.2 microg/mL) for 1 day to induce apoptosis. Detection of apoptotic bodies under differential interference contrast microscopy was combined with immunocytochemistry for BrdU, proliferating cell nuclear antigen (PCNA) or for various markers of retinal cell differentiation. Despite the large number of BrdU- and PCNA-labelled cells in the tissue, the vast majority of the cells that underwent apoptosis were postmitotic cells which have left the mitotic cycle 3-4 days before. However, these cells were not labelled with antibodies to calretinin, calbindin, rhodopsin or to a Muller glial cell marker, suggesting that these are early postmitotic neurons. We suggest that during migration and initial differentiation, the apoptotic machinery is blocked by suppressor proteins, thus allowing recent postmitotic cells to find their final positions and differentiate while protected from apoptosis.

Apoptosis in developing retinal tissue.

The mechanisms of apoptosis are strongly dependent on cell-cell interactions typical of organized tissues. Experimental studies of apoptosis using a histotypical preparation of retinal explants are reported in the present article. We found that various characteristics of apoptosis are selectively associated with retinal cell death depending on cell type, stage of maturation, and means of induction of apoptosis. Among these were: (1) the requirements of protein synthesis; (2) the role of cAMP; (3) the expression of certain apoptosis-associated proteins; and (4) the sensitivity to excitotoxicity, modulation of protein phosphatases and calcium mobilization. Dividing cells undergo apoptosis in response to several inducers in specific phases of the cell cycle, and in distinct regions within their pathway of interkinetic nuclear migration. Recent post-mitotic cells are selectively sensitive to apoptosis induced by blockade of protein synthesis, while both proliferating and differentiated cells are more resistant. We also studied the association of several proteins, some of which play critical roles in the cell cycle, with both differentiation and apoptosis in the retinal tissue. Detection of cell cycle markers did not support the hypothesis that retinal cells re-enter the cell cycle on their pathway to apoptosis, although some proteins associated with cell proliferation re-appeared in degenerating cells. The transcription factors c-Jun, c-Fos and c-Myc were found associated with apoptosis in retinal cells, but their sub-cellular location in apoptotic bodies is not consistent with their canonical functions in the control of gene expression. The bifunctional redox factor/AP endonuclease Ref-1 and the transcription factor Max are associated with progressive cell differentiation, and both are down-regulated during cell death in the retina. The data suggest that Ref-1 and Max may normally function as negative modulators of retinal apoptosis. The results indicate that nuclear exclusion of transcription factors and other important control proteins is a hallmark of retinal apoptosis. Histotypical explants may be a choice preparation for the experimental analysis of the mechanisms of apoptosis, in the context both of cell-cell interactions and of the dynamic behavior of developing cells within the organized retinal tissue.

Developmentally regulated release of intraretinal neurotrophic factors in vitro.

The effects of conditioned media either from aggregates or from explants of embryonic chick retinae and of recombinant neurotrophins were tested upon the survival in vitro of ganglion cells in dissociated cell cultures from the retina of newborn rats. Ganglion cells were identified by the detection of retrogradely transported horseradish peroxidase injected bilaterally into the superior colliculus. Conditioned media increased significantly the survival of ganglion cells after 2 days in culture, at a wide range of plating densities, and had no effect upon adhesion of rat retinal cells. Media conditioned by cell ensembles from chick retinae from embryonic day 8 (E8) to E16 had neurotrophic effects. Release of neurotrophic activity peaked at E10 E12, irrespective of the numbers of cells or total concentration of protein in the conditioned media. The active molecules were non-dialyzable and were released either in the presence or in the absence of fetal calf serum. The neurotrophic activity was abolished by trypsinization, and recovered by salting-out with 25 75% ammonium sulfate. NT-4, BDNF and, to a lesser extent, NT-3, increased the survival of ganglion cells in our assay, while NGF had no effect. The data show that chick retinal cells release soluble trophic proteins according to a developmentally regulated pattern. These neurotrophic factors may be involved in local competitive interactions that help control naturally occurring neuron death among ganglion cells of the vertebrate retina.

Death in a dish: controls of apoptosis within the developing retinal tissue.

Studies of programmed cell death in the developing retina in vitro are currently reviewed. The results of inhibiting protein synthesis in retinal explants indicate two mechanisms of apoptosis. One mechanism depends on the synthesis of positive modulators ('killer proteins'), while a distinct, latent mechanism appears to be continuously blocked by negative modulators. Extracellular modulators of apoptosis include the neurotrophic factors NT-4 and BDNF, while glutamate may have either a positive or a negative modulatory action on apoptosis. Several protein kinases selectively modulate apoptosis in distinct retinal layers. Calcium and nitric oxide were also shown to affect apoptosis in the developing retinal tissue. The protein c-Jun was found associated with apoptosis in various circumstances, while p53 seems to be selectively expressed in some instances of apoptosis. The results indicate that the sensitivity of each retinal cell to apoptosis is controlled by multiple, interactive, cell type- and context-specific mechanisms. Apoptosis in the retina depends on a critical interplay of extracellular signals delivered through neurotrophic factors, neurotransmitters and neuromodulators, several signal transduction pathways, and the expression of a variety of genes.

Death in dish: controls of apoptosis within the developing retinal tissue

Studies of programmed cell death in the developing retina in vitro are currently reviewed. The results of inhibiting protein synthesis in retinal explants indicate two mechanisms of apoptosis. One mechanism depends on the synthesis of positive modulators ('killer proteins'), while a distinct, latent mechanism appears to be continuously blocked by negative modulators. Extracellular modulators of apoptosis include the neurotrophic factors NT-4 and BDNF, while glutamate may have either a positive or a negative modulatory action on apoptosis. Several protein kinases selectively modulate apoptosis in distinct retinal layers. Calcium and nitric oxide were also shown to affect apoptosis in the developing retianl tissue. The protein c-Jun was found associated with apoptosis in various circumstances, while p53 seems to be selectively expressed in some instances of apoptosis. The results indicate that the sensitivity of each retinal cell to apoptosis is controlled by multiple, interactive, cell type- and context-specific mechanisms. Apoptosis in the retina depends on a critical interplay of extracellular signals delivered through neurotrophic factors, neurotransmitters and neuromodulators, several signal transduction pathways, and the expression of a variety of genes.

Contrasting effects of protein synthesis inhibition and of cyclic AMP on apoptosis in the developing retina.

The role of protein synthesis in apoptosis was investigated in the retina of developing rats. In the neonatal retina, a ganglion cell layer, containing neurons with long, centrally projecting axons, is separated from an immature neuroblastic layer by a plexiform layer. This trilaminar pattern subsequently evolves to five alternating cell and plexiform layers that constitute the mature retina and a wave of programmed neuron death sweeps through the layers. Apoptosis due to axon damage was found in ganglion cells of retinal explants within 2 days in vitro and was prevented by inhibition of protein synthesis. Simultaneously, protein synthesis blockade induced apoptosis among the undamaged cells of the neuroblastic layer, which could be selectively prevented by an increase in intracellular cyclic AMP. Both the prevention and the induction of apoptosis among ganglion cells or neuroblastic cells, respectively, occurred after inhibition of protein synthesis in vivo. The results show the coexistence of two mechanisms of apoptosis within the organized retinal tissue. One mechanism is triggered in ganglion cells by direct damage and depends on the synthesis of proteins acting as positive modulators of apoptosis. A distinct, latent mechanism is found among immature neuroblasts and may be repressed by continuously synthesized negative modulators, or by an increase in intracellular cyclic AMP.

Apoptosis in the developing retina: paradoxical effects of protein synthesis inhibition.

Cell death by apoptosis is usually characterized as an active process that requires protein and RNA synthesis. The requirement of protein synthesis for the degeneration of ganglion cells and other cell types was studied in neural retinae explanted from the eyes of newborn rats. Ganglion cells were detected by the presence of retrogradely transported horseradish peroxidase injected into the superior colliculus. Apoptotic cells were recognized by their condensed and deeply stained chromatin. The data show that the death of ganglion cells, whose axons are damaged when preparing the explants, is blocked or delayed by protein synthesis inhibitors. In contrast, the blockade of protein synthesis produced cell death with apoptotic morphology in the neuroblastic layer of the same retinae. The results suggest the operation in the developing retina of both a program of apoptosis dependent on the synthesis of killer proteins, and a latent mechanism of apoptosis that is normally blocked by repressor proteins.

Apoptosis in the developing retina: paradoxical effects of protein synthesis inhibition

Cell death by apoptosis is usually characterized as an active process that requires protein and RNA synthesis. The requirement of protein synthesis for the degeneration of ganglion cells and other cell types was studied in neural retinae explanted from the eyes of newborn rats. Ganglion cells were detected by the presence of retrogradely transported horseradish peroxidase injected into the superior colliculus. Apoptotic cells were recognized by their condensed and deeply stained chromatin. The data show that the death of ganglion cells, whose axons are damaged when preparing the explants, is blocked or delayed by protein synthesis inhibitors. In contrast, the blockade of protein synthesis produced cell death with apoptotic morphology in the neuroblastic layer of the same retinae. The results suggest the operation in the developing retina of both a program of apoptosis dependent on the synthesis of killer proteins, and a latent mechanism of apoptosis that is normally blocked by repressor proteins.

Intraretinal neurotrophic activity prevents the degeneration of ganglion cells in retinal explants.

The degeneration of ganglion cells was studied in neural retina explanted from the eyes of newborn rats. The ganglion cells were detected by the presence of retrogradely transported horseradish peroxidase injected into the superior colliculus. The time course of cell death among the axotomized ganglion cells in the explants was similar to that found in vivo after axotomy in neonatal rats. The effect of culture media conditioned with retinal cells from either newborn rats or chick embryos was tested on the survival of ganglion cells in the explants. Both conditioned media increased 2- to 3-fold the survival of rat retinal ganglion cells after 2 days in culture. The data show that soluble trophic factors released by retinae of distinct species can influence the survival of ganglion cells within their histotypic microenvironment.

Intraretinal neurotrophic activity prevents the degeneration of ganglion cells in retinal explants

The degeneration of ganglion cells was studied in neural retina explanted from the eyes of newborn rats. The ganglion cells were detected by the presence of retrogradely transported horseradish peroxidase injected into the superior colliculus. The time course of cell death among the axotomized ganglion cells in the explants was similar to that found in vivo after axotomy in neonatal rats. The effect of culture media conditioned with retinal cells from either newborn rats or chick embryos was tested on the survival of ganglion cells in the explants. Both conditioned media increased 2- to 3-fold the survival of rat retinal ganglion cells after 2 days in culture. The data show that soluble trophic factors released by retinae of distinct species can influence the survival of ganglion cells within their histotypic microenvironment

Evidence for a developmentally regulated neurotrophic activity from chick retina affecting the survival of retinal ganglion cells from newborn rats.

The effect of conditioned medium from aggregates of chick embryo retinal cells was tested on the in vitro survival of retinal ganglion cells from newborn rats. Ganglion cells were identified by the detection of retrogradely transported horseradish peroxidase injected bilaterally into the superior colliculus. Culture medium conditioned with chick embryo retinae was tested on monolayers of rat retinal cells with plating densities ranging from 0.5 to 4 x 10(5) cells/cm2. In all cases the conditioned medium significantly increased the survival of ganglion cells after 2 days in culture. Conditioned media from embryonic days 8 to 16 (E8 to E16) presented neurotrophic activity, with the greatest effect occurring at E10-E12. The conditioned medium had no effect on the adhesion of rat retinal cells. The data suggest that chick retinal cells produces soluble trophic factors which can influence the survival of rat retinal ganglion cells in vitro. Furthermore, the release of this neurotrophic activity by chick retina seems to be developmentally regulated.