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S F Hunter

Publications and source records attributed to S F Hunter.

6 recordsLinked to original sources
PubMed1991

O-2A glial progenitors from mature brain respond to CNS neuronal cell line-derived growth factors.

During development, myelin-forming oligodendrocytes and type 2 astrocytes are believed to arise from bipotential (O-2A) glial progenitors. Previously we found that conditioned medium (CM) from the B104 rat CNS neuronal cell line promotes growth of neonatal rat O-2A progenitors in serum-free culture conditions with subsequent increases in differentiated progeny. We now report that O-2A progenitors are present in mature rat brains and that this CM promotes the growth, motility, and bipolar morphology of these cells from 30- and 65-day-old rat brains, as shown by quantitative studies using double immunostaining and [3H]thymidine-autoradiography. In addition, the growth-promoting action of B104 CM is not neutralized by antibodies to platelet-derived growth factor, a proposed progenitor mitogen. Subsequent to the proliferation of these O-2A progenitors, increases in oligodendrocytes and type 2 astrocytes occur. These data suggest a novel therapeutic strategy for some demyelinating diseases, e.g., multiple sclerosis, where there is a deficit in oligodendrocytes. Although it has been proposed by others that mature brain O-2A progenitors are less proliferative and thereby incapable of adequately replenishing lost oligodendrocytes in these diseases, we present in vitro evidence for continued response of mature brain O-2A progenitors to this neuronal cell line-derived mitogen.

Animals↗
PubMed1991

Isolation of arteriolar microvessels and culture of smooth muscle cells from cerebral cortex of guinea pig.

Published methods for the isolation of cerebral microvessels primarily yield terminal resistance vessels and capillary networks, not the more proximal, subpial penetrating arterioles desired for certain studies. We report a novel method for isolating microvessels from the cerebral cortex of a single guinea-pig brain that yields large arteriolar complexes that are up to 50% intact. Instead of using homogenization to disperse brain parenchyma, we digested cortical fragments with trypsin, gently dispersed the parenchyma mechanically, and recovered microvascular complexes by sieving. Phase-contrast and electron microscopy showed primary (penetrating) arterioles, secondary arterioles, and capillary networks that frequently were in continuity as intact microvascular units. Culture of microvascular cells was carried out by enzymatic dissociation followed by an overnight incubation in a recovery medium at 4 degrees C before plating onto fibronectin-modified surfaces. Viability of isolated cells was demonstrated by good cell attachment and prompt proliferation that resulted in confluent cultures after 10 days. Confluent secondary cultures demonstrated characteristic features of smooth muscle cells, including a "hill-and-valley" growth pattern and expression of alpha-actin. Less than 1% of cells were endothelial or astrocytic cells by immunocytochemical and morphologic criteria. Ultrastructural studies demonstrated evidence of a synthetic phenotype of smooth muscle cell and absence of a significant number of fibroblasts. This method demonstrates that viable smooth muscle cells from the cerebral parenchymal microvasculature can be isolated in bulk quantities for study in vitro.

Actins↗
PubMed1990

Growth factor responses of enriched bipotential glial progenitors.

Responses of oligodendrocyte/type 2 astrocyte (O-2A) glial progenitors from neonatal rat brains to different growth factors were studied by a new, serum-free method. Enriched tertiary cultures of O-2A progenitors were produced after 6-7 days in vitro using the growth-promoting factors from the B104 CNS neuronal cell line, heparin, and mechanical separation. These cultures contained about 75-90% A2B5+ cells with less than 10% type 1 astrocytes, and the yield was 4.4 x 10(5) cells/brain. B104 conditioned medium (CM) factors increased both O-2A progenitor number and [3H]thymidine-labeling indices after three days. However, type 1 astrocyte CM was required for continued survival of enriched progenitors beyond 1 day in tertiary culture. Platelet-derived growth factor (PDGF) and glia maturation factor also showed growth-promoting action, but were less effective than B104 CM at tested doses. PDGF-neutralizing antibodies had no effect on progenitor survival or response to B104 CM factors. Thus, type 1 astrocyte-derived PDGF was not required for this response, B104 CM is not likely to contain PDGF, and B104 CM factors act directly on O-2A progenitors. Fibroblast growth factor, transforming growth factor beta, interleukin 2, epidermal growth factor, and triiodothyronine showed no growth-promoting activity; moreover, interleukin 2, epidermal growth factor, transforming growth factor beta, and 0.5% fetal bovine serum inhibited B104 CM action. Enriched progenitors exhibited bipotentiality by slowly differentiating into oligodendrocytes in serum-free medium, whereas culture in 10% fetal bovine serum increased type 2 astrocytes. Thus, this new method selects or produces progenitors which are similar to those from mature brains.

Animals↗
PubMed1989

Bipotential glial progenitors are targets of neuronal cell line-derived growth factors.

The growth-promoting activity of conditioned medium (CM) from the B104 CNS neuronal cell line was studied in glial cultures from neonatal rat brain. This CM at 33% (v/v; 8-12 micrograms protein/ml) produced large numbers of oligodendrocytes and multipolar glial progenitors after an 8 to 12-day treatment. At all times studied, cells of the oligodendrocyte/type 2 astrocyte (O-2A) lineage were increased due to CM-treatment, while type 1 astrocytes, microglia, and other cell types were not. Furthermore, we observed a large decrease in the percentage of oligodendrocytes in the O-2A lineage, suggesting a delay in differentiation of the progenitors. By 8 days in vitro (DIV), dose-dependent increases in numbers of galactocerebroside (GalC)-positive cells (oligodendrocytes) and A2B5-positive cells (immature oligodendrocytes and glial progenitors) occurred. In contrast, at 4 DIV only A2B5-positive cells were increased in a dose-dependent manner. The latter cells can differentiate primarily into oligodendrocytes or type 2 astrocytes depending on the culture conditions. Complement lysis studies confirmed that the A2B5-positive, but not the GalC-positive, population at 4 DIV was required for increases in oligodendrocytes to occur by 8 DIV. The [3H]thymidine labeling index of the A2B5-positive population also increased in response to CM in a dose-dependent manner, but the GalC-positive labeling index showed only small increases at 4 DIV and none at later times. Our results suggest that the delayed differentiation coupled with the selective stimulation of the bipotential glial progenitors produces the large increases in numbers of oligodendrocytes observed at 8-12 DIV.

Animals↗
PubMed1988

CNS neuronal cell line-derived factors regulate gliogenesis in neonatal rat brain cultures.

Recent studies suggest that heterotypic cell-cell interactions influence gliogenesis in the developing rat central nervous system. CNS neuron-derived factors have been hypothesized to exist, and several have been identified and partially characterized which affect the number of oligodendrocytes in vitro. In order to study further the role of neurons in gliogenesis, we have used serum-free culture conditions, the B104 CNS neuronal cell line as a source of soluble factors, and dissociated neonatal rat brain cells as a source of glial cells. We have analyzed the response of the glial cells to serum-free B104 conditioned medium using morphological, immunocytochemical, autoradiographic, and enzymatic methods. Dose-dependent increases in the number of morphologically identified oligodendrocytes occur in response to this conditioned medium. Galactocerebroside (GalC) is a specific marker for oligodendrocytes, and the A2B5 antigen marks bipotential glial progenitor cells and their progeny: immature oligodendrocytes and type 2 astrocytes. In the presence of conditioned medium, the number of cells expressing GalC and/or A2B5 antigen increases over time when measured at 4, 8, and 12 days in vitro. A significantly weaker effect is seen if serum is also present. Since the vast majority of A2B5-positive cells in conditioned medium treated cultures lack glial fibrillary acidic protein (GFA), indicative of type 2 astrocytes, they represent glial progenitors and immature oligodendrocytes. Double immunostaining combined with autoradiography suggests that the latter cell types are the target cells for the oligodendrocyte-promoting activity. In addition, the conditioned medium markedly increases 2',3' cyclic nucleotide 3'-phosphodiesterase (an oligodendrocyte marker) and to a lesser extent enhances glutamine synthetase activity (an astrocyte marker). Type 1 astrocytes are also more morphologically differentiated in this condition, and their percentage is decreased simultaneously. Conditioned medium from other donor neural cells either has no activity or is much less effective than B104 conditioned medium. The active factors are soluble, sensitive to both trypsin and 100 degrees C treatment for 20 min, and appear to be 30-100 kilodaltons by stirred cell ultrafiltration. In summary, we have identified a potent source of growth-stimulating factors that produce increased numbers of glial progenitor cells and oligodendrocytes; the same conditioned medium also appears to inhibit type 1 astrocyte proliferation.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗