Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes

Background

The aim of this study was to investigate gene transfer to human umbilical cord blood (CB) CD34⁺/CD38^low and NOD/SCID repopulating cells using oncoretroviral vectors and to compare the transduction efficiency using three different viral envelopes.

Methods

CB cells were transduced on Retronectin using an MSCV‐based vector with the gene for GFP (MGIN), which was packaged into three different cell lines giving different envelopes: PG13‐MGIN (GALV), 293GPG‐MGIN (VSV‐G) or AM12‐MGIN (amphotropic).

Results

Sorted CD34⁺/CD38^low cells were efficiently transduced after 3 days of cytokine stimulation and the percentage of GFP‐positive cells was 61.8±6.6% (PG13‐MGIN), 26.9±3.5% (293GPG‐MGIN), and 39.3±4.8% (AM12‐MGIN). For transplantation experiments, CD34⁺ cells were pre‐stimulated for 2 days before transduction on Retronectin preloaded with vector and with the addition of 1/10th volume of viral supernatant on day 3. On day 4, the expanded equivalent of 2.5×10⁵ cells was injected into irradiated NOD/SCID mice. All three pseudotypes transduced NOD/SCID repopulating cells (SRCs) equally well in the presence of serum, but engraftment was reduced when compared with freshly thawed cells. Simultaneous transduction with all three vector pseudotypes increased the gene transfer efficiency to SRCs but engraftment was significantly impaired. There were difficulties in producing amphotropic vectors at high titers in serum‐free medium and transduction of CD34⁺ cells using VSV‐G‐pseudotyped vectors under serum‐free conditions was very inefficient. In contrast, transduction with PG13‐MGIN under serum‐free conditions resulted in the maintenance of SRCs during transduction, high levels of engraftment (29.3±6.6%), and efficient gene transfer to SRCs (46.2±4.8%).

Conclusions

The best conditions for transduction and engraftment of CB SRCs were obtained with GALV‐pseudotyped vectors using serum‐free conditions. Copyright © 2002 John Wiley & Sons, Ltd.

     

Neurite outgrowth from hippocampal neurons is promoted by choroid plexus ependymal cells <Emphasis Type="Italic">in vitro</Emphasis>

Choroid plexus ependymal cells (CPECs) were known to promote axonal growth when choroid plexus is grafted into the adult rat spinal cord. The present study was carried out to examine whether CPECs promote axonal outgrowth from neurons derived from the CNS in vitro. Hippocampal neurons were cocultured on CPEC monolayers. After 24 h, neurite extension was evaluated using various parameters in comparison with cultures grown on poly-L-lysine (PLL)-coated plates and cocultures grown on astrocyte monolayers. The primary neurite length and total neurite length were longest in the cocultures with CPECs. The number of primary neurites and the number of branches were larger in the cultures with CPECs than in the cultures on PLL-coated plates, but almost the same as in the cocultures with astrocytes. Next, we examined whether the neurite extension-promoting effect occurring within 24 h is due primarily to contact with the CPECs or to factors secreted by CPECs into the culture medium. The CPEC monolayers were killed by ethanol fixation, and neurons cultured on them. The neurons extended long neurites with elaborate branching, as in the case of cocultures grown on living CPECs. On the other hand, CPEC-conditioned medium exhibited less promoting effect on neurite outgrowth from hippocampal neurons. These results indicate that CPECs have a capacity to promote neurite outgrowth from CNS neurons in vitro, and that surface plasma membrane-bound components of CPECs strongly contribute to the enhancement of neurite outgrowth in the present coculture system.     

Metabolism and Release of Glutamate in Cerebellar Granule Cells Cocultured with Astrocytes from Cerebellum or Cerebral Cortex

Cerebellar granule cells were cocultured with astrocytes from either cerebral cortex or cerebellum in two different systems. In one system the cells were plated next to each other only sharing the culture medium (separated cocultures) and in the other system the granule cells were plated on top of a preformed layer of astrocytes (sandwich cocultures). Using astrocytes from cerebellum, granule cells developed morphologically and functionally showing a characteristic high activity of the glutamate synthesizing enzyme aspartate aminotransferase (AAT) as well as a high stimulus-coupled transmitter release regardless of the culture system, i.e., granule cells could grow on top of cerebellar astrocytes as well as next to these cells. In the case of cerebral cortex astrocytes it was found that cerebellar granule cells did not develop (11% survival) when seeded on top of these astrocytes. This was indicated by the morphological appearance of the cultures as well as by a negligible difference between the AAT activity in sandwich cocultures and astrocytes cultured alone. On the other hand, granule cells in separated cocultures with cerebral cortex astrocytes exhibited a normal morphology and a high activity of AAT as well as a large stimulus-coupled transmitter release. Cerebellar and cortical astrocytes expressed the astrocyte specific enzyme glutamine synthetase in a glucocorticoid-inducible form regardless of the culture system. The results show that under conditions of direct contact between granule cells and astrocytes, regional specificity exists with regard to neuron-glia contacts. This specificity does not seem to involve soluble factors present in the culture medium because in separated cocultures the cerebellar granule cells developed normally regardless of the regional origin of the astrocytes.     

Astrocytes Enhance the Invasion Potential of Glioblastoma Stem-Like Cells

Glioblastomas (GBMs) are characterized as highly invasive; the contribution of GBM stem-like cells (GSCs) to the invasive phenotype, however, has not been completely defined. Towards this end, we have defined the invasion potential of CD133+ GSCs and their differentiated CD133− counterparts grown under standard in vitro conditions and in co-culture with astrocytes. Using a trans-well assay, astrocytes or astrocyte conditioned media in the bottom chamber significantly increased the invasion of GSCs yet had no effect on CD133− cells. In addition, a monolayer invasion assay showed that the GSCs invaded farther into an astrocyte monolayer than their differentiated progeny. Gene expression profiles were generated from two GSC lines grown in trans-well culture with astrocytes in the bottom chamber or directly in contact with astrocyte monolayers. In each co-culture model, genes whose expression was commonly increased in both GSC lines involved cell movement and included a number of genes that have been previously associated with tumor cell invasion. Similar gene expression modifications were not detected in CD133− cells co-cultured under the same conditions with astrocytes. Finally, evaluation of the secretome of astrocytes grown in monolayer identified a number of chemokines and cytokines associated with tumor cell invasion. These data suggest that astrocytes enhance the invasion of CD133+ GSCs and provide additional support for a critical role of brain microenvironment in the regulation of GBM biology.     

Purity,cell viability,expression of GFAP and bystin in astrocytes cultured by different procedures

Primary astrocyte cultures are the most commonly used in vitro model for neurobiological studies. We speculated that different protocols might induce differences not only in the percentage of astrocytes but also in their biological characteristics. In this study, we investigated the effects of four major protocols on the purity of astrocytes, cell viability, expression of glial fibrillary acidic protein (GFAP) and bystin of cultured astrocytes using MTT assay, immunocytochemical staining, and Western blot analysis. We demonstrated that the purity of astrocytes (98.9%) generated by the subculture (SC) procedure is significantly higher than those generated by primary culture (PC), shaken once culture (SK‐1) or shaken twice culture (SK‐2). We also showed that expressions of GFAP and bystin in astrocytes that are purified by the SK‐2 or SK‐1 procedures are significantly higher than those in astrocytes prepared by PC or SC. In addition, astrocytes cultured by SK‐2 or SK‐1 have a higher level of cell viabilities at most time points after ischemia compared with astrocytes cultured by PC or SC. These suggested that physical stimulation induced by “shaken” or culture operation might be able to activate astrocytes and implied that different procedures induce differences not only in the purity but also in the biological characteristics of astrocytes, such as the percentage of activated astrocytes, GFAP, and bystin expressions and responses to ischemia. A more detailed analysis about the effect of “culture protocol factor” on the biological characteristics of astrocytes is absolutely needed. J. Cell. Biochem. 109: 30–37, 2010. © 2009 Wiley‐Liss, Inc.     

Time course study of L-tyrosine aminotransferase induction in rat liver cell lines

The enhancement of L-tyrosine aminotransferase activity by dexamethasone, an exclusive function of the liver, was serially measured at different passages of eight rat liver epithelial cell lines initiated and continuously grown in either a serum-supplemented medium or a serum-free medium. The enzyme basal activity was found to be 5.4 ± 1.8 mU for cell lines in serum and 6.8 ± 3.4 mU for cell lines without serum. Under the influence of dexamethasone (10^–6 mol/l for 5 hours) this basal level could be increased up to 2.9 fold in the presence of serum and 2.5 fold in its absence when investigations were carried out at early passages. During the following subcultures the induction ratio gradually declined and scarcely any induction could be detected after the 15th passage for cells grown in serum and after the 25th passage for cell lines grown without serum.Abbreviations SFM serum-free medium - SSM serum-supplemented medium - TAT L-tyrosine aminotransferase M.F. is a recipient of a government scholarship grant from the Grand Duchd de Luxembourg.     

Effect of Fatty Acids Isolated from Edible Oils Like Mustard,Linseed or Coconut on Astrocytes Maturation

The omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA, 22:6n-3) has been previously shown to facilitate some of the vital functions of astrocytes. Since some dietary oils contain alpha-linolenic acid (ALA, 18:3n-3), which is a precursor of DHA, we examined their effect on astrocyte development. Fatty acids (FAs) were isolated from commonly used oils and their compositions were determined by GLC. FAs from three oils, viz. coconut, mustard and linseed were studied for their effect on astrocyte morphology. Parallel studies were conducted with FAs from the same oils after heating for 72 h. Unlike coconut oil, FAs from mustard and linseed, both heated and raw, caused significant morphogenesis of astrocytes in culture. ss-AR binding was also substantially increased in astrocytes treated with FAs from raw mustard and linseed oils as compared to astrocytes grown in normal medium. The expression profile of the isoforms of GFAP showed that astrocyte maturation by FAs of mustard and linseed oil was associated with appearance of acidic variants of GFAP and disappearance of some neutral isoforms similar to that observed in cultures grown in serum containing medium or in the presence of DHA. Taken together, the study highlights the contribution of specific dietary oils in facilitating astrocyte development that can have potential impact on human health.     

Tat 蛋白的PTD区段促进GFP蛋白进入骨髓瘤细胞SP2/0

随着生物工程技术的迅速发展 ,多肽与蛋白质类药物的增长速度相当可观 ,可是这些药物常因受到各种因素的影响而疗效偏低 ,其中生物膜的屏障作用是主要因素之一。近年来发现一种来源于人类免疫缺陷病毒ＨＩＶ 1Ｔａｔ(Ｔｒａｎｓ ａｃｔｉｖａ ｔｏｒ)蛋白的蛋白功能区 ,称之为ＰＴＤ区段 (Ｐｒｏｔｅｉｎｔｒａｎｓｄｕｃｔｉｏｎｄｏｍａｉｎ ,ＹＧＲＫＫＲＲＱＲＲＲ)的〔1 ,2〕,能够有效引导肽段或者蛋白质进入细胞 ,具有蛋白传送的功能〔3〕。 1988年Ｍａｕｒｉｃｅ和Ｐａｕｌ发现Ｔａｔ蛋白能够穿过细胞膜〔4〕 ;1994年Ｓｔｅｐｈｅｎ…     

A Modified In vitro Method to Obtain Pure Astrocyte Cultures Induced from Mouse Hippocampal Neural Stem Cells Using Clonal Expansion

The aim of the present study was to produce astrocyte cultures of high purity from mouse hippocampal neural stem cells and to compare their in vitro properties with those isolated from enriched mixed glial cultures prepared from mouse hippocampus, which are commonly contaminated by microglia. We produced primary cultures of newborn mouse hippocampal neural stem cells, which have the potential to differentiate into astrocytes, neurons, and oligodendrocytes. We produced monoclonal neural stem cell colonies by limiting dilution. We induced astrocyte differentiation by plating the colonies on poly-l-lysine and culturing them in induction medium consisting of minimum essential medium/F12 supplemented with 10% fetal bovine serum and 100 ng/ml ciliary neurotrophic factor. We then further purified the cells by differential adherence and shaking at a constant temperature, followed by a second round of limiting dilution. Immunocytochemistry for glial fibrillary acidic protein showed that our method yielded 99.4 ± 0.5% pure astrocytes, whereas traditionally enriched mixed glial cultures yielded 94.2 ± 2% pure astrocytes. Induced cells resembled primary astrocyte cultures in functional properties such as cell proliferation rates and lack of tumorigenicity and p53, and expression of epidermal growth factor receptor, bystin, and nitric oxygen synthase. Our novel method of culture and purification of neural stem cells can therefore be used routinely for the primary culture of highly purified astrocytes from mouse hippocampus.     

Modulation of beta-adrenergic response in rat brain astrocytes by serum and hormones

Purified astrocyte cultures from neonatal rat cerebrum respond to isoproterenol, a beta-adrenergic agonist, with a transient rise in cAMP production. This astroglial property was regulated by serum, a chemically defined medium (serum-free medium plus hydrocortisone, putrescine, prostaglandin F2 alpha, insulin, and fibroblast growth factor) and epidermal growth factor. Compared to astrocytes grown in serum-supplemented medium, astrocytes grown in the chemically defined medium were nonresponsive to isoproterenol stimulation, and this difference did not appear to be due to selection of a subpopulation of cells by either medium. The data suggest that a decreased beta-adrenergic receptor number and an increased degradation of cAMP may account for the reduced response to beta-adrenergic stimulation. The nonresponsive state of astrocytes in the defined medium was reversible when the medium was replaced with serum-supplemented medium. An active substance(s) in serum was responsible for restoring the responsiveness of astrocytes. Each of the five components of the defined medium had little effect by itself; however, together they acted synergistically to desensitize astrocytes to beta-adrenergic stimulation. On the other hand, epidermal growth factor, a potent mitogen for astrocytes, was very competent by itself in reducing the cAMP response of astrocytes to beta-adrenergic stimulation. Thus purified astrocytes grown in the chemically defined medium appear to be a good model for the study of hormonal interactions and of serum factors which may modulate the beta-adrenergic response.     

Expression of the green fluorescent protein carried by Autographa Californica multiple nucleopolyhedrovirus in insect cell lines

Summary A recombinant AcMNPV containing the green fluorescent protein (gfp) gene under the polyhedrin promoter (polh) was used to investigate the expression of the gfp gene as well as the production of recombinant extracellular virus in 14 continuous insect cell lines, including Heliothis virescens (BCIRL-HV-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), Spodoptera exigua (BCIRL/AMCY-Se-E1 and BCIRL/AMCY-Se-E5), Bombyx mori (BMN), Sf9 (a clone of IPLB-SF21), and five cell line clones of BCIRL-HV-AM1. The susceptibility of the cell lines to the recombinant virus (AcMNPV.GFP) was ascertained by calculating the mean percentage number of green light-emitting cells as well as by TCID₅₀ titration of extracellular virus with fluorescence as a sign of infection. Of the 14 cell lines tested, all were permissive with varying degrees to Ac-MNPV.GFP, except BCIRL-HV-AMCL2 and BCIRL-HZ-AM1, both grown in serum-containing medium, and BMN, grown in serum-free medium, which were nonpermissive to the virus. Except for BCIRL/AMCY-Se-E1, IPLB-SF21, and four of the five BCIRL-HV-AM1 clones, all the other cell lines (BCIRL-HV-AM1, BCIRL-AG-AM1, TN-CL1, Se-E5, and Sf9) expressed detectable levels of GFP by 48 h postinoculation. The BCIRL/AMCY-Se-E1 and IPLB-SF21 cells, grown in serum-free medium (Ex-Cell 401), expressed detectable levels of GFP at 72 h postinoculation. By contrast, in BCIRL/AMCY-Se-E1 in serum-containing medium (Ex-Cell 401+10% FBS [fetal bovine serum]), GFP was detected at 48 h postinoculation. Furthermore, TN-CL1 cells produced the largest mean percentage number of fluorescent (76.6%) cells in both serum-containing and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. The mean extracellular virus (ECV) production at 120 h postinoculation was highest in BCIRL/AMCY-Se-E5 cells grown in Ex-Cell 401+10% FBS (37.8×10⁶ TCID₅₀/ml) followed by BCIRL-HV-AM1 in TC199-MK (33.4×10⁶ TCID₅₀/ml). Only the BCIRL-HV-AMCL3 clone produced any substantial level of ECV at 120 h postinoculation (16.9×10⁶ TCID₅₀/ml). However, there was no significant correlation between ECV production and the mean percentage number of fluorescent cells. This study provides further information on the susceptibility of 14 insect cell lines to a recombinant AcMNPV containing the green fluorescent protein gene. This information might avail researchers with information to facilitate decisions as to what other cell lines are available for in vitro studies of the gfp gene.     

Direct cloning of astrocytes from primary culture without previous immortalization

Summary In primary cultures, much evidence shows the existence of different subtypes of astrocytes that are not all identified. One methodology for studying these subtypes can be their cloning. The present investigation shows a method for a direct cloning of astrocytes without previous immortalization. Astrocytes from the cerebral cortex of newborn rats were cultured, purified by shaking, and harvested by trypsinization. One single astrocyte was plated in a small volume of a homemade cloning medium. After getting a colony, successive platings were made using larger and larger vessels, up to 60-mm-diameter petri dishes. Then, subcultures were made. The yield of the cloning was similar to that of common eukaryotic cell clonings. All along the cloning procedure, the cells were positively immunostained with anti-glial fibrillary acidic protein antibodies. Cloned cells from some batches were spindle-shaped, looking like fibroblasts. Nevertheless, they were immunostained with anti-glial fibrillary acidic protein antibodies, unlike true fibroblasts. These spindle-shaped astrocytes were compared to cells from an astrocytoma cell line that had the same shape. The growth pattern of the astrocytoma cells was different from that of the astrocytes cloned from the primary cultures. All the types of studied cells contained glycogen. On the basis of the criteria of morphology, of glial fibrillary acidic protein immunolabeling, and of glycogen synthesis, the cloned cells kept the characteristics of astrocytes. This study shows that it is perfectly possible to get clones of astrocytes from one astrocyte without previous immortalization, giving thus a convenient material for the study of astrocyte biology.     

Short-term culture of myeloid leukemic cells allows efficient transduction by adenoviral vectors

BACKGROUND: Ex vivo gene therapy of acute myeloid leukemia (AML) requires efficient transduction of leukemic cells. Recombinant adenovirus has been reported to be a poorly efficient vector in leukemic cells. We investigated leukemic cell culture as a possible method of improving the efficacy of this vector. METHODS: Leukemic cell lines and primary cultured AML cells were incubated with adenoviral vectors carrying GFP, LacZ, or IL-12 cDNA. Transduction efficiency was evaluated by measuring adenoviral genome copy number and transgene expression in leukemic cells. The expression of the coxsackie/adenovirus receptor (CAR), CD29, CD49e, and CD51/61 was measured, as was the effect of blocking integrin on adenoviral transduction. RESULTS: Increasing the multiplicity of infection (MOI) to 300 plaque-forming units per cell enhanced transduction of leukemic cell lines and to a lesser degree of AML cells. Analysis of adenoviral genome copy per cell showed only a partial correlation between gene transfer efficiency and transgene expression. Culture of AML cells for 3 days prior to adenoviral transduction increased both adenoviral copy number per cell and the percentage of transgene-expressing cells. CD29, CD49e, and CD51/61 but not CAR expression increased in cultured AML cells between days 0 and 3 and integrin-blocking experiments showed inhibition of transduction in two of four AML samples tested. CONCLUSIONS: Efficient ex vivo gene transfer in primary cultured AML cells can be achieved by short-term culture of leukemic cells prior to gene transfer with adenoviral vectors at a high MOI. This effect appears to be at least partially mediated by enhanced integrin expression.     

HIV-1 TAT-mediated protein transduction and subcellular localization using novel expression vectors

Several novel prokaryotic and eukaryotic expression vectors were constructed for protein transduction and subcellular localization. These vectors employed an N-terminal stretch of 11 basic amino acid residues (47-57) from the human immunodeficiency virus type 1 (HIV-1) TAT protein transduction domain (PTD) for protein translocation and cellular localization. The vectors also contained a six-histidine (His(6)) tag at the N- or C-terminus for convenient purification and detection, and a multiple cloning site for easy insertion of foreign genes. Some heterologous genes including HSV-TK, Bcl-rambo, Smac/DIABLO and GFP were fused in-frame to TAT PTD and successfully overexpressed in Escherichia coli. The purified TAT-GFP fusion protein was able to transduce into the mammalian cells and was found to locate mainly in the cytosol when exogenously added to the cell culture medium. However, using a transfection system, mammalian-expressed TAT-GFP predominantly displayed a nuclear localization and nucleolar accumulation in mammalian cell lines. This discrepancy implies that the exact subcellular localization of transduced protein may depend on cell type, the nature of imported proteins and delivery approach. Taken together, our results demonstrate that a TAT PTD length of 11 amino acids was sufficient to confer protein internalization and its subsequent cellular localization. These novel properties allow these vectors to be useful for studying protein transduction and nuclear import.     

Pharmacological and Functional Heterogeneity of Astrocytes: Regional Differences in Phospholipase C Stimulation by Neuromediators

Carbamylcholine stimulated phospholipase C activity in astrocytes in primary culture from the mesencephalon but not from the striatum or cerebral cortex of the mouse embryo. An alpha 1-adrenergic-mediated response was observed in all astrocyte populations. 2-Chloroadenosine potentiated the alpha 1-adrenergic response in mesencephalic and striatal astrocytes but not in cortical astrocytes. It also stimulated the carbamylcholine-evoked response in mesencephalic astrocytes. Through cell-cell cooperation, 2-chloroadenosine potentiated the neuronal carbamylcholine-evoked activation of phospholipase C in homotopic cocultures (neuro-glial) from the striatum but not in homotopic cocultures (neuro-glial) from the cerebral cortex or in heterotopic cocultures (cortical astrocytes-striatal neurons; striatal astrocytes-cortical neurons.     

Gene transfer of human TCR in primary murine T cells is improved by pseudo-typing with amphotropic and ecotropic envelopes

BACKGROUND: T cell receptor (TCR) gene therapy represents an attractive anti-cancer treatment but requires further optimization of its efficacy and safety in clinically relevant models, such as those using a tumor antigen and TCR of human origin. Currently, however, there is no consensus as to what protocol is most optimal for retroviral human TCR gene transfer into primary murine T cells, most notably with respect to virus pseudo-type. METHODS: Primary murine T cells were transduced, expanded and subsequently tested for transgene expression, proliferation and antigen-specific function. To this end, murine leukemia virus (MLV) retroviruses were produced upon transfection of various packaging cells with genes encoding either green fluorescent protein (GFP) or TCRalphabeta specific for human melanoma antigen gp100(280-288) and the helper elements GAG/POL and ENV. Next to viral pseudotyping, the following parameters were studied: T cell densities; T cell activation; the amounts of IL-2 and the source of serum used to supplement medium. RESULTS: The pseudo-type of virus produced by packaging cells critically determines T cell transduction efficiencies. In fact, MLV-A and MLV-E pseudo-typed viruses derived from a co-culture of Phoenix-A and 293T cells resulted in T cell transduction efficiencies that were two-fold higher than those based on retroviruses expressing either VSV-G, GALV, MLV-A or MLV-E envelopes. In addition, T cell densities during transduction were inversely related to transduction efficiencies. Further optimization resulted in transduction efficiencies of over 90% for GFP, and 68% for both a murine and a human (i.e. murinized) TCR. Importantly, TCR-transduced T cells proliferate (i.e. showing a log increase in cell number in a few days) and show antigen-specific function. CONCLUSIONS: We set up a quick and versatile method to genetically modify primary murine T cells based on transient production of TCR-positive retroviruses, and show that retroviral gene transfer of a human TCR into primary murine T cells is critically improved by viral pseudo-typing with both MLV-A and MLV-E envelopes.     

Proteoglycans of reactive rat cortical astrocyte cultures: Abundance of N-unsubstituted glucosamine-enriched heparan sulfate

“Reactive” astrocytes and other glial cells in the injured CNS produce an altered extracellular matrix (ECM) that influences neuronal regeneration. We have profiled the glycosaminoglycan (GAG) component of proteoglycans (PGs) produced by reactive neonatal rat cortical astrocytes, and have quantified their neurite-outgrowth inhibitory activity. PGs extracted from cell layers and medium were fractionated on DEAE-Sephacel with a gradient of NaCl from 0.15 to 1.0 M. Monosaccharide analysis of the major peaks eluting at 0.6 M NaCl indicated an excess of GlcNH₂ to GalNH₂, suggesting an approximate HS/CS ratio of 6.2 in the cell layer and 4.2 in the medium. Chondroitinase ABC-generated disaccharide analysis of cell and medium PGs showed a > 5-fold excess of chondroitin 4-sulfate over chondroitin 6-sulfate. Heparin lyase-generated disaccharides characteristic of the highly sulfated S-domain regions within HS were more abundant in cell layer than medium-derived PGs. Cell layer and medium HS disaccharides contained ~ 20% and ~ 40% N-unsubstituted glucosamine respectively, which is normally rare in HS isolated from most tissues. NGF-stimulated neurite outgrowth assays using NS-1 (PC12) neuronal cells on adsorbed substrata of PGs isolated from reactive astrocyte medium showed pronounced inhibition of neurite outgrowth, and aggregation of NS-1 cells. Cell layer PGs from DEAE-Sephacel pooled fractions having high charge density permitted greater NGF-stimulated outgrowth than PGs with lower charge density. Our results indicate the synthesis of both inhibitory and permissive PGs by activated astrocytes that may correlate with sulfation patterns and HS/CS ratios.