Purification and characterization of adult oligodendrocyte precursor cells from the rat optic nerve - PubMed (original) (raw)

Purification and characterization of adult oligodendrocyte precursor cells from the rat optic nerve

J Shi et al. J Neurosci. 1998.

Abstract

Oligodendrocyte precursor cells (OPCs) persist in substantial numbers in the adult brain in a quiescent state suggesting that they may provide a source of new oligodendrocytes after injury. To determine whether adult OPCs have the capacity to divide rapidly, we have developed a method to highly purify OPCs from adult optic nerve and have directly compared their properties with their perinatal counterparts. When cultured in platelet-derived growth factor (PDGF), an astrocyte-derived mitogen, perinatal OPCs divided approximately once per day, whereas adult OPCs divided only once every 3 or 4 d. The proliferation rate of adult OPCs was not increased by addition of fibroblast growth factor (FGF) or of the neuregulin glial growth factor 2 (GGF2), two mitogens that are normally produced by retinal ganglion cells. cAMP elevation has been shown previously to be essential for Schwann cells to survive and divide in response to GGF2 and other mitogens. Similarly we found that when cAMP levels were elevated, GGF2 alone was sufficient to induce perinatal OPCs to divide slowly, approximately once every 4 d, but adult OPCs still did not divide. When PDGF was combined with GGF2 and cAMP elevation, however, the adult OPCs began to divide rapidly. These findings indicate that adult OPCs are intrinsically different than perinatal OPCs. They are not senescent cells, however, because they retain the capacity to divide rapidly. Thus, after demyelinating injuries, enhanced axonal release of GGF2 or a related neuregulin might collaborate with astrocyte-derived PDGF to induce rapid division of adult OPCs.

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Figures

Fig. 1.

Fig. 1.

The differentiation of adult OPC cells.A, B, Immunofluorescence micrographs of purified adult OPCs that were labeled after 4 d of culture with a monoclonal anti-GC antibody (A) or a polyclonal GFAP antiserum (B). When cultured in serum-free medium containing insulin and CNTF (A), nearly all cells differentiated into GC+ oligodendrocytes. When cultured in medium containing 10% fetal calf serum (B), nearly all cells differentiated into GFAP+ type-2 astrocytes. Scale bar, 50 μm.

Fig. 2.

Fig. 2.

Hoffman micrograph of an adult OPC clone. Purified cells were plated at clonal density and cultured for 21 d in serum-free medium containing PDGF, NT-3, CNTF, and insulin. Adult OPCs have a bipolar morphology that is indistinguishable from perinatal OPCs. Scale bar, 100 μm.

Fig. 3.

Fig. 3.

The proliferation rate of adult and perinatal OPCs in culture. Purified adult (P60) (A, C) and perinatal (P8) (B, D) OPCs were cultured at clonal density in serum-free medium containing PDGF and insulin. The number of cells per clone was determined after 4 d (A, B) and 8 d (C,D) of culture. Under these culture conditions, which lack T3, most of the clones consisted predominantly of OPCs. Note that the average size of adult OPC clones is significantly smaller than is that of the perinatal clones.

Fig. 4.

Fig. 4.

Comparison of the proliferation rate of adult and perinatal OPCs in vitro and in vivo. A, OPCs were cultured in serum-free medium containing PDGF, NT-3, CNTF, and insulin for 4 d before a 90 min incubation with BrdU (10 μ

m

). The percentage of cells that incorporated BrdU was determined by immunostaining. Values are mean ± SEM (n = 3 coverslips).B, BrdU was injected intraperitoneally into adult and P8 rats. After 90 min, OPCs were purified from the optic nerves of the injected rats and cultured for 1 hr before BrdU staining.

Fig. 5.

Fig. 5.

Comparison of the proliferation rate of adult and perinatal OPCs in cryosections. A, B, Immunofluorescence micrographs of optic nerve cryosections from P8 (A) and adult (B) optic nerves. The cryosections were obtained from optic nerves that were fixed 90 min after an intraperitoneal injection of BrdU and double labeled with NG-2 (green) and BrdU (red) antibodies. Note that in the P8 section, there are many NG-2+ cells that are also BrdU+. In contrast, in the adult section, none of the NG-2+ cells are BrdU+. Scale bar, 50 μm.

Fig. 6.

Fig. 6.

Effects of T3 on the rate of oligodendrocyte generation by OPCs. Purified P60 (A) and P8 (B) OPCs were cultured at clonal density in serum-free medium containing PDGF, NT-3, CNTF, and insulin in the presence (solid circles) and absence (open circles) of T3. The percentages of clones containing predominantly oligodendrocytes (>50% of cells) were counted after 4, 6, 8, and 12 d of culture. All values are mean ± SEM. T3 strongly enhanced the rate of oligodendrocyte generation from both P60 and P8 OPCs.

Fig. 7.

Fig. 7.

Effects of GGF2 on adult OPCs. Purified P60 and P8 OPCs were cultured for 8 d at clonal density in serum-free medium containing PDGF, GGF2,IBMX (0.1 m

m

), and forskolin (5 μ

m

), as indicated. A, The division rate of P8 and P60 was calculated from the clone size. B, The rate of oligodendrocyte generation by P60 OPCs was determined by the percentage of clones that primarily consisted of oligodendrocytes. GGF2 enhances the rate of proliferation of P60 OPCs and inhibits their differentiation into oligodendrocytes. All values are mean ± SEM.FORSK, Forskolin; IBMX, isobutylmethylxanthine.

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