Growth and differentiation properties of O-2A progenitors purified from rat cerebral hemispheres

Abstract

We have used the monoclonal antibody A2B5 (which binds to subclasses of surface gangliosides) to select glial precursor cells from postnatal rat brain and compare their properties in culture with those of the bipotential O-2A progenitor cells of newborn optic nerve. Two methods, fluorescence-activated cell sorting (FACS) and differential adhesion, resulted in greater than 90% enrichment in A2B5-positive bipolar cells and multipolar cells with short processes. These cells expressed vimentin and reacted with yet another antibody (NSP4), which binds to O-2A progenitor cells of optic nerve. The 2-10% of the remaining cells consisted of type 1 astrocytes and/or microglial cells. When maintained in defined medium for 3 days, 28-40% of A2B5-positive cells incorporated thymidine, while most other cells became differentiated into galactocerebroside-positive oligodendrocytes. In the presence of 10% fetal calf serum for 3 days, over 50% of the cells developed a stellate phenotype and expressed GFAP, characteristic of type 2 astrocytes. This phenotypic plasticity of the A2B5 positive cells was also observed in clones derived from single cells grown on a layer of type 1 astrocytes. Thus, A2B5-positive cells from cerebrum are O-2A progenitors that can generate O-2A lineage cells. The effects of the two growth factors, insulin and platelet derived growth factor (PDGF) (which is synthesized by type 1 astrocytes), were tested on cerebrum O-2A progenitors. PDGF induced a doubling of the percentage of A2B5-positive cells incorporating thymidine during a 20-hr pulse and a large increase (up to 40-fold) of the progenitor population over 3 days. The largest number of O-2A lineage cells was obtained when purified progenitors were grown in the presence of PDGF and insulin. Thus, A2B5-positive glial cells from cerebrum overall behave as the O-2A progenitors of optic nerve, but they more readily divide than differentiate, as if they were at an earlier stage along the O-2A lineage pathway.