Morphological changes do not reflect biochemical and functional differentiation in OLN-93 oligodendroglial cells (original) (raw)

Long-Term Culture of Purified Postnatal Oligodendrocyte Precursor Cells

Journal of Cell Biology, 2000

Oligodendrocytes myelinate axons in the vertebrate central nervous system (CNS). They develop from precursor cells (OPCs), some of which persist in the adult CNS. Adult OPCs differ in many of their properties from OPCs in the developing CNS. In this study we have purified OPCs from postnatal rat optic nerve and cultured them in serum-free medium containing platelet-derived growth factor (PDGF), the main mitogen for OPCs, but in the absence of thyroid hormone in order to inhibit their differentiation into oligodendrocytes. We find that many of the cells continue to proliferate for more than a year and progressively acquire a number of the characteristics of OPCs isolated from adult optic nerve. These findings suggest that OPCs have an intrinsic maturation program that progressively changes the cell's phenotype over many months. When we culture the postnatal OPCs in the same conditions but with the addition of basic fibroblast growth factor (bFGF), the cells acquire these mature c...

Isolation, culture, and characterization of adult rat oligodendrocytes

Journal of Neuroscience Research, 1990

Investigation into CNS demyelinating diseases, which usually occur in adults, can be facilitated by the use of a good in vitro model. We have established a methodology whereby oligodendrocytes from adult rat CNS can be cultured in vitro, and we have characterized these cultures morphologically and immunologically. Approximately 1 g of spinal cord and brainstem per adult rat was removed and dissociated mechanically and enzymatically. After filtration of the white matter homogenate, myelin was removed by 0.9 M sucrose density centrifugation. The cells were further purified by centrifugation through a 0.3%/4% discontinuous gradient of bovine serum albumin (BSA). The pellet was resuspended and placed in an untreated 6-well culture dish overnight to allow the astrocytes to attach. The non-adherent cells were replated on poly-I-lysine-treated coverslips. Approximately 8.25 X lo5 cells were recovered per animal. The adult oligodendrocytes initially appeared as rounded cell bodies, but after 2-5 days in vitro (DIV), the oligodendrocytes extended 6-10 thick processes. A membrane sheath between these processes was immunostainable with either anti-galactocerebroside (GC), anti-04, anti-myelin basic protein (MBP), or anti-2'3' cyclic nucleotide 3' phosphohydrolase (CNPase) and was also evident in scanning EM. Older cultures (up to 60 DIV) maintained whorls of myelin and transmission EM revealed a major dense line distance of approximately 103 A with up to 11 concentric layers of membrane. Immunologically, the adult oligodendrocytes are G C + , 0 4 + , MBP+, CNPase+, and GFAP-. The method described will allow adult rat oligodendrocytes to be isolated and maintained in culture; these cultures retain the characteristics of differentiated adult oligodendrocytes.

Phenotypic and cell cycle properties of human oligodendrocytes in vitro

Brain research, 1995

The remyelination, albeit limited, which occurs at the lesion sites in the central nervous in multiple sclerosis has been attributed to both myelin production by previously myelinating cells and to precursor cells which mature into myelin-producing cells. Oligodendrocyte (OL) number may be increased at the periphery of the lesions. In this study, we assessed the state of maturation and cell cycle-dependent properties of OLs derived from surgically resected adult human cerebral cortex specimens. In 6-day-old OL cultures, a small proportion of cells (14.1 + 3.5%: range 4-24%) expressed an immature phenotype, defined as A007+:myelin basic protein (MBP)-negative. Using lai~k of statin expression as an index of cells exiting the GO phase of the cell cycle, we observed that 4.6 + 1.6% of A007 ÷ cells, but only rare MBP ÷ cells (0.4 + 1.8%) were non-reactive with the anti-statin antibody, $44. The proportion of non-statin-reactive cells was not affected by treatment with basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) or insulin-like growth factor (IGF). The oligodendrocytes did not incorporate BrdU during a 48-h pulse and did not immunoreact with Ki-67 antibody. In 4-week-old cultures, we found that all A007 ÷ cells were also MBP ÷ and that 99.5 + 0.7% were statin-positive. Exposing 4-week-old OLs to conditions of serum deprivation or to 1,000 units/ml of recombinant human TNF-/3 for 4 days induced nuclear fragmentation in a high proportion ( > 70%) of cells, as measured by a TUNEL technique; in these cultures, a similarly high proportion of cells were non-immunoreactive with anti-statin antibody. Our results suggest that a small number of phenotypic 'pre-oligodendrocytes' can be derived from the adult human CNS and that a proportion of these cells have exited the GO phase of the cell cycle. Attempt at cell cycling, however, could reflect abortive mitosis and activation of programmed cell death.

Reappraisal of Human HOG and MO3.13 Cell Lines as a Model to Study Oligodendrocyte Functioning

Cells

Myelination of neuronal axons is essential for proper brain functioning and requires mature myelinating oligodendrocytes (myOLs). The human OL cell lines HOG and MO3.13 have been widely used as in vitro models to study OL (dys) functioning. Here we applied a number of protocols aimed at differentiating HOG and MO3.13 cells into myOLs. However, none of the differentiation protocols led to increased expression of terminal OL differentiation or myelin-sheath formation markers. Surprisingly, the applied protocols did cause changes in the expression of markers for early OLs, neurons, astrocytes and Schwann cells. Furthermore, we noticed that mRNA expression levels in HOG and MO3.13 cells may be affected by the density of the cultured cells. Finally, HOG and MO3.13 co-cultured with human neuronal SH-SY5Y cells did not show myelin formation under several pro-OL-differentiation and pro-myelinating conditions. Together, our results illustrate the difficulty of inducing maturation of HOG and ...

Isolation and purification of primary oligodendrocyte precursors

2001

Oligodendrocytes are the myelinating cells of the central nervous system, and are avidly studied by people interested in neural cell lineages, myelination, multiple sclerosis, and oligodendroglioma. As with other neural cells, the ability to obtain pure populations is critical for performing experiments on many aspects of their biology. Purification protocols for oligodendrocytes have been based on sedimentation techniques, to exploit the fact that their density is different from that of other central nervous system cells.

Serum contains inducers and repressors of oligodendrocyte differentiation

Journal of Neuroscience Research, 1988

An important stage in oligodendrocyte development is the expression of galactocerebroside (GC), the major glycolipid in myelin. Although oligodendrocyte cell liieage and differentiation in vitro have been the object of many studies, to date there is sparse information on the regulation of GC expression in oligodendrocytes already committed to be positive for GC. We report here that GC expression in these cells is controlled by three serum factors. Two of these, possibly a lipoprotein and a mucoprotein, increase GC levels, whereas the third, probably a glycoprotein, exerts an inhibitory effect. The developmental increase of GC in postnatal rat brain cerebral cultures and its induction by serum factors are reversible phenomena. The isolation of the GCregulatory factors would allow experimental manipulation of impaired GC expression by differentiated oligodendrocytes.

An oligodendrocyte precursor cell line from rat optic nerve

Brain Research, 1992

We have established permanent cell lines from the optic nerve of the rat with a temperature sensitive immortalizing oncogene (Simian Virus 40 large T-antigen carrying both the tsA58 and U19 mutations), The oncogene was transduced into primary cultures via a replication deficient retrovirus, and infected cells were selected with the antiobiotic G418. A clonal cell line (tsU19-5) displayed some properties of oligodendrocyte precursors: it proliferated, bound the monoclonal antibody A2B5 (which recognizes minor ganglioside species), and expressed the intermediate filament vimentin and the enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) at 33°C (the permissive temperature for the oncogene). At 39°C (the non-permissive temperature), some cells had the potential to differentiate further, and expressed several oligodendrocyte specific components: galactocerebroside, myelin basic protein, proteolipid protein and CNP. These results suggest that conditional oncogenes can establish neural precursor cell lines which are still capable of differentiation in vitro.

Growth requirements in vitro of oligodendrocyte cell lines and neonatal rat brain oligodendrocytes

Proceedings of the National Academy of Sciences of the United States of America, 1986

I have defined the basic requirements for the proliferation of cell lines expressing oligodendrocyte properties and for the survival of galactocerebroside-positive oligodendrocytes derived from neonatal rat brains. Conventional serum-containing medium can be replaced by 01 medium, a chemically defined medium supplemented with insulin, transferrin, sodium selenite, and biotin. Thyroid hormone is not required. When cells are plated directly into 01 medium, the substratum has to be modified by precoating with polylysine and adding fibronectin to the medium prior to the cells. Both cell lines and brain cells can be subcultured numerous times in 01 medium without initial culture in serum-containing medium. Brain cultures can be maintained in 01 medium for several months and contain a significantly higher percentage of mature oligodendrocytes, a lower number of astrocytes, and no fibroblasts as compared to cells maintained in serum-containing medium.