Supported phospholipid bilayers as a platform for neural progenitor cell culture.

Supported phospholipid bilayers constitute a biomimetic platform for cell behavior studies and a new approach to the design of cell culture substrates. Phosphocholine bilayers are resistant to cell attachment, but can be functionalized with bioactive molecules to promote specific cell interactions. Here, we explore phosphocholine bilayers, functionalized with the laminin-derived IKVAV pentamer, as substrates for attachment, growth, and differentiation of neural progenitor cells (AHPs). By varying peptide concentration (0-10%), we discovered a strongly nonlinear relationship between cell attachment and IKVAV concentration, with a threshold of 1% IKVAV required for attachment, and saturation in cell binding at 3% IKVAV. This behavior, together with the 10-fold reduction in cell attachment when using a jumbled peptide sequence, gives evidence for a specific interaction between IKVAV and its AHP cell-surface receptor. After 8 days in culture, the peptide-functionalized bilayers promoted a high degree of cell cluster formation. This is in contrast to the predominant monolayer growth, observed for these cells on the standard laminin coated growth substrates. The peptide-functionalized bilayer did not induce differentiation levels over those observed for the laminin coated substrates. These results are promising in that peptide-functionalized bilayers can allow attachment and growth of stem cells without induction of differentiation.

Reactive astrogliosis induces astrocytic differentiation of adult neural stem/progenitor cells in vitro.

Neural stem cells reside in defined areas of the adult mammalian brain, including the dentate gyrus of the hippocampus. Rat neural stem/progenitor cells (NSPCs) isolated from this region retain their multipotency in vitro and in vivo after grafting into the adult brain. Recent studies have shown that endogenous or grafted NSPCs are activated after an injury and migrate toward lesioned areas. In these areas, reactive astrocytes are present and secrete numerous molecules and growth factors; however, it is not currently known whether reactive astrocytes can influence the lineage selection of NSPCs. We investigated whether reactive astrocytes could affect the differentiation, proliferation, and survival of adult NSPCs by modelling astrogliosis in vitro, using mechanical lesion of primary astrocytes. Initially, it was found that conditioned medium from lesioned astrocytes induced astrocytic differentiation of NSPCs without affecting neuronal or oligodendrocytic differentiation. In addition, NSPCs in coculture with lesioned astrocytes also displayed increased astrocytic differentiation and some of these NSPC-derived astrocytes participated in glial scar formation in vitro. When proliferation and survival of NSPCs were analyzed, no differential effects were observed between lesioned and nonlesioned astrocytes. To investigate the molecular mechanisms of the astrocyte-inducing activity, the expression of two potent inducers of astroglial differentiation, ciliary neurotrophic factor and leukemia inhibitory factor, was analyzed by Western blot and shown to be up-regulated in conditioned medium from lesioned astrocytes. These results demonstrate that lesioned astrocytes can induce astroglial differentiation of NSPCs and provide a mechanism for astroglial differentiation of these cells following brain injury.

Demonstration of multiple novel glycoforms of the stem cell survival factor CCg.

We have investigated the presence of different glycoforms of cystatin C secreted by adult hippocampal rat-derived stem/progenitor cells (AHPs) into conditioned medium. A glycosylated form of cystatin C (CCg) has been identified previously in conditioned medium from AHPs as an autocrine/paracrine cofactor. Fibroblast growth factor-2 (FGF2) requires cooperation with CCg to support AHP survival at low density in vitro. The purpose of the present study was to investigate further if cystatin C consists of one glycoform or if several different glycoforms are secreted by AHPs in vitro. The presence of the glycoforms was studied using enzymatic deglycosylation in conjunction with gel electrophoresis and Western blotting. The glycoforms of cystatin C were isolated with a combination of gel electrophoresis and electroelution, yielding the intact glycoforms in liquid phase before enzymatic deglycosylation. Our results revealed several novel glycoforms, in contrast to previous publication. The results suggest that N- and O-linked glycans with sialic acid are attached to cystatin C. Furthermore, we have demonstrated that all glycoforms are present in conditioned medium after only 48 hr of culturing and that all nestin-positive AHPs are immunopositive against cystatin C. These findings suggest secretion of the glycoforms by cultured AHPs.

Selective introduction of antisense oligonucleotides into single adult CNS progenitor cells using electroporation demonstrates the requirement of STAT3 activation for CNTF-induced gliogenesis.

We have developed a novel method in which antisense DNA is selectively electroporated into individual adult neural progenitor cells. By electroporation of antisense oligonucleotides against signal transducer and activator of transcription 3 (STAT3) we demonstrate that ciliary neurotrophic factor (CNTF) is an instructive signal for astroglial type 2 cell fate specifically mediated via activation of STAT3. Activation of the mitogen-activated protein kinase (MAPK) signaling pathway induced only a transient increase in glial fibrillary acidic protein (GFAP) expression, and inhibition of this signaling pathway did not block the induction by CNTF of glial differentiation in progenitor cells. In addition we show that microelectroporation is a new powerful method for introducing antisense agents into single cells in complex cellular networks.

Regulation of G-PROTEIN mRNA abundancy and cAMP accumulation after long-term opioid incubation in primary cultures of astroglia from the rat cerebral cortex.

Primary astroglial cultures were incubated with delta (10(-6) M DPDPE) or kappa (10(-5) M U-50,488H) receptor agonists for 5 days. Thereafter, the acute inhibitory actions of delta or kappa receptor agonists on forskolin stimulated cAMP accumulation were assayed. The G alpha s, G alpha i-1 and G alpha i-2 mRNA levels were quantified after 5 days of either delta or kappa receptor agonist treatment using a solution hybridization, RNase protection assay. Pronounced effects were observed after 5 days of kappa receptor agonist [10(-5) M U-50,488H] incubation. This treatment resulted in an attenuation in the acute inhibitory action of delta and kappa receptor agonists. Furthermore, a decreased stimulatory action of forskolin was seen. Similar effects were also seen after delta receptor stimulation. We also investigated the effects after 24 h and 3 days of incubation with the kappa receptor agonist (10(-5) M) U-50,488H. The 24 h incubation resulted in a decreased sensitivity to the acute inhibitory action of delta and kappa receptor agonists in the astroglial cultures. This effect was further accentuated after the 3 days of incubation with 10(-5) M U-50,488H. No significant change was seen in the basal accumulation of cAMP after incubation with the kappa agonist U-50,488H. However, after 5 days of incubation with the delta agonist DPDPE, a significantly increased basal accumulation of cAMP was seen in the astroglial cultures. After 5 days of delta or kappa agonist incubation, an increase in G alpha s mRNA level and a decrease in G alpha i-2 mRNA level was seen compared with controls. No statistically significant alterations in the amount of G alpha i-1 mRNA were seen. The data obtained in the present study indicate that the effects of long-term opioid treatment alters the sensitivity of glial cell opioid receptors. Furthermore, long term opioid treatment induces alterations in glial G-protein mRNA levels.

Altered amounts of G-protein mRNA and cAMP accumulation after long-term opioid receptor stimulation of neurons in primary culture from the rat cerebral cortex.

Primary neuronal enriched cultures were incubated with mu (morphine, 10(-5) M), delta (DPDPE, 10(-6) M) and kappa (U-50,488H, 10(-5) M) receptor agonists for 5 days, respectively. Thereafter the acute inhibitory actions of mu, delta or kappa receptor agonists on forskolin stimulated cAMP accumulation was assayed. The effect of long term opioid treatment on the steady-state level of G-protein mRNA (G alpha s, G alpha i-1 and G alpha i-2) was analyzed using an RNAase protection hybridization assay. Incubation for 5 days with kappa receptor agonist resulted in an attenuated ability to decrease the accumulation of cAMP by kappa receptors, as well as mu and delta receptors, which was also observed after 5 days of incubation with the delta receptor agonist. Furthermore, the adenylate cyclase responsiveness to forskolin stimulation was markedly reduced in cultures treated with either delta or kappa receptor agonists. Five days of incubation with kappa receptor agonist resulted in an increase in the levels of G alpha s and G alpha i-2 mRNAs. No effects on the amounts of G alpha s mRNA, G alpha i-1 mRNA or G alpha i-2 mRNA were detected after 5 days of delta receptor stimulation. On the other hand, 5 days of mu receptor stimulation decreased the amounts of G alpha s, G alpha i-1 and G alpha i-2 mRNA. Incubation with kappa receptor agonist for 24 h resulted in a significant decrease in the forskolin-stimulated accumulation of cAMP. The stimulatory effect of forskolin was further decreased after 3 days incubation with kappa receptor agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Delta and kappa opiate receptors in primary astroglial cultures. Part II: Receptor sets in cultures from various brain regions and interactions with beta-receptor activated cyclic AMP.

In a previous paper, delta and kappa opiate receptors were shown to be co-localized on the same cell in enriched primary cultures of astroglia from neonatal rat cerebral cortex. Activation of the receptors inhibited adenylate cyclase. In this work, the presence of opiate receptors was investigated in astroglial primary cultures from neonatal rat striatum and brain stem. Cyclic adenosine 3',5'-monophosphate accumulation was quantified in the presence of different opioid receptor ligands after stimulation of the cyclic adenosine 3',5'-monophosphate system with forskolin. Morphine was used as a mu receptor agonist. [D-Ala2, D-Leu5]-enkephalin or [D-Pen2, D-Pen5]-enkephalin were used as delta receptor agonists and dynorphin 1-13 or U-50,488H were used as kappa receptor agonists. Specific antagonists for the respective receptors were used. After striatum or brain stem cultures had been incubated in 10(-9)-10(-5) M of each [D-Ala2, D-Leu5]-enkephalin, [D-Pen2, D-Pen5]-enkephalin and Dynorphin 1-13 or U-50,488H, dose related inhibitions of the 10(-5) M forskolin stimulated cyclic adenosine 3',5'-monophosphate accumulation were observed. The changes were reversed to the forskolin-induced control level in the presence of the respective antagonists. 10(-9)-10(-5) M morphine did not significantly change the forskolin-induced accumulation of cyclic adenosine 3',5'-monophosphate in the cultures studied. Furthermore, cultures from cerebral cortex, striatum or brain stem were incubated with isoproterenol alone or together with morphine or [D-Ala2, D-Leu5]-enkephalin.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic elevation of cAMP levels induces changes in the adenylate cyclase system, opiate receptor sensitivity and levels of Gs-mRNA in cultured neurons.

Neurons in primary culture were treated (5 days) with the adenylate cyclase stimulator 10(-5) M forskolin. The basal adenylate cyclase was decreased by 57%. The acute stimulatory effect of forskolin was down-regulated by 48%. The inhibitory effects of the 3 opiate receptor agonists (mu, delta and kappa) were partly abolished. The abundance of mRNA encoding the stimulatory G-protein (Gs) was decreased prominently. The data indicate that the cAMP system in neuronal cells in primary culture is under dynamic regulation, possibly including altered Gs-protein gene expression. Furthermore, long-term forskolin treatment might induce increased proliferation in susceptible neural blast cells.

Mu and delta opiate receptors in neuronal and astroglial primary cultures from various regions of the brain--coupling with adenylate cyclase, localisation on the same neurones and association with dopamine (D1) receptor adenylate cyclase.

Primary cultures, enriched in neurones or astroglial cells, from three phylogenetically different regions of the brain of the rat, the cerebral cortex, the striatum and the brain stem, were used to investigate the presence of opiate receptors, coupled to adenylate cyclase. Morphine was used as a mu-receptor agonist and [D-Ala2, D-Leu5]-enkephalin (DADLE) was used as a delta-receptor agonist. In the neuronal cultures, both ligands inhibited the prostaglandin (PG)E1-stimulated intracellular accumulation of cyclic AMP dose-dependently, with the most prominent effects seen in the cultures of striatum and with DADLE being more potent than morphine. The opiate receptor antagonist, naloxone reversed the effects. Morphine and DADLE, added together, inhibited the PGE1-stimulated accumulation of cyclic AMP, less than the sum of the effects of each drug. Therefore, it might be that these opioid receptors are localized together on the same neurone. Striatal neurones contained dopamine receptors coupled to cyclic AMP, as second messenger. It was shown that the D1 (dopamine) receptor-stimulated activity of adenylate cyclase was inhibited by the mu and delta opioid receptor ligands. Thus, interactions at the level of adenylate cyclase seem to exist between D1, mu and delta opiate receptors. In the astroglial enriched cultures, DADLE inhibited the PGE1-induced accumulation of cyclic AMP, however, with a less prominent effect in the brain stem cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of glutathione in primary neuronal and astroglial cultures from rat cerebral cortex and brain stem.

The concentration of the tripeptide glutathione (GSH) was measured in primary cultures of neurons and astroglial cells from rat cerebral cortex and brain stem. The concentration of GSH was found to be approximately 20 nmol/mg protein in the neuronal culture from the cerebral cortex and ca. 40 nmol/mg protein in the neuronal brain stem cultures. A GSH concentration of approximately 20 nmol/mg was observed in the astrocyte cultures from both brain regions. The possibility to increase the GSH concentration was tested by incubating the cultures in the presence of the GSH precursor gamma-glutamylcysteine (gamma-GC). In the cultured astrocytes gamma-GC produced a dose-dependent increase in GSH. A similar increase was observed in the neuronal cultures, but this effect failed to reach statistical significance.

Delta and kappa opiate receptors in primary astroglial cultures from rat cerebral cortex.

The effects of mu, delta, and kappa receptor-agonists on forskolin stimulated cyclic adenosine-3',5'-monophosphate (cAMP) formation were examined in astroglial enriched primary cultures from the cerebral cortex of newborn rats. Intracellular cAMP accumulation was quantified by radioimmunoassay. Morphine was used as a mu-receptor agonist, D-Ala-D-Leu-Enkephalin (DADLE) as a delta-receptor agonist and dynorphin 1-13 (Dyn) as a kappa-receptor agonist. Basal cAMP levels were unaffected by either the opiate agonists or the antagonists used. In the presence of the cAMP stimulator forskolin, morphine had no significant effect on the cytoplasmic cAMP levels. DADLE caused a dose related inhibition of the forskolin stimulated cAMP accumulation. The effects of this delta receptor stimulation was blocked with the selective antagonist ICI 174.864. In the presence of Dyn, the forskolin stimulated cAMP accumulation was inhibited in a dose related manner. This kappa receptor stimulation was blocked with the selective antagonist MR 2266. Co-administration of DADLE and Dyn resulted in a non additive inhibition of the forskolin stimulated accumulation of cAMP. These findings indicate that astroglial enriched cultures from the cerebral cortex of rats express delta and kappa-receptors co-localized on the same population of cells, and that these receptors are inhibitory coupled to adenylate cyclase.

Opiate receptors in neuronal primary cultures.

The occurrence and characteristics of mu-, delta- and kappa-receptors were studied as effects of the respective agonists on forskolin-stimulated accumulation of cAMP in neuronal enriched primary cultures from the cerebral cortex of foetal rats. Morphine or [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO) were used as mu-receptor agonists. [D-Ala2,D-Leu5]-Enkephalin (DADLE) or [D-Pen2,D-Pen5]-enkephalin (DPDPE) were used as delta-receptor agonists and dynorphin 1-13 (Dyn) or U-50,488H were used as kappa-receptor agonists. In the presence of 10(-8)-10(-5) M morphine or 10(-8)-10(-5) M DAGO, there was a dose-related inhibition of the 10(-5) M forskolin-stimulated accumulation of cAMP. The inhibitory action of morphine or DAGO was reversed by naloxone. In the presence of 10(-9)-10(-6) M DADLE or 10(-9)-10(-6) M DPDPE, there was also a dose-related inhibition of the forskolin-stimulated accumulation of cAMP and a similar result was obtained in the presence of 10(-9)-10(-5) M Dyn or 10(-9)-10(-5) M U-50,488 H. These findings indicate that neurones from the cerebral cortex in culture express mu-, delta- and kappa-receptors, that inhibit the forskolin-stimulated accumulation of cAMP. Administration of 10(-5) M morphine and 10(-6) M DADLE or 10(-6) M DPDPE together, resulted in a non-additive inhibition of the forskolin-stimulated accumulation of cAMP, indicating the presence of both mu- and delta-receptors on the same population of cells.