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. 1998 Mar 31;95(7):3996–4001. doi: 10.1073/pnas.95.7.3996

Figure 2.

Figure 2

A2B5+ cells derived from E13.5 spinal cord are multipotent GRP cells. A2B5+ cells were immunopurified from E13.5 rat spinal cord and grown as clones in medium supplemented with PDGF + bFGF. (A) A2B5+ cells did not express markers of neuroblasts, neurons, astrocytes, or oligodendrocytes. (B) After 5 days of growth in PDGF + bFGF, cloning dishes were switched to medium containing either FCS or PDGF + T3 for 5 days. In dishes of primary clones switched to FCS, all clones generated A2B5+ and A2B5− astrocytes. Clones grown in PDGF + T3 contained oligodendrocytes and GRP cells but few astrocytes. The total of 132 clones in FCS and 81 clones in PDGF + T3 represents the sum of three independent experiments. To examine whether GRP cells were capable of extensive self-renewal without loss of differentation potential, five primary clones were recloned in PDGF + bFGF. Three to five clones were selected randomly from each set of secondary clones and recloned a third time. Dishes were switched to medium containing either FCS or PDGF + T3, and in each condition, three descendant clones of each initial clone were chosen for analysis thus yielding the 15 clones analyzed in each condition. As shown, the ability of the freshly cloned GRPs to differentiate into oligodendrocytes and two types of astrocytes was retained fully, even after tertiary cloning. (C–F) A single primary A2B5+ clone that was recloned into separate dishes and induced to differentiate for 5 days in the presence of PDGF + T3 (C and D) or FCS (E and F), after which cultures were immunolabeled. A2B5 staining (D and F) is red, anti-GalC staining (C) is green, and anti-GFAP staining (E) is blue. A clone derived from a single A2B5 cell can differentiate into A2B5+GFAP+ astrocytes (arrows in E and F), A2B5−/GFAP+ astrocytes (unlabeled nonprocess-bearing astrocytes in E, and A2B5− GalC+ oligodendrocytes (C). This recloning experiment was repeated three times, from three independent dissections, with identical results. (Bar = 25 μm for C and D, 50 μm for E and F).