Redox state is a central modulator of the balance between self-renewal and differentiation in a dividing glial precursor cell

Abstract

We have discovered that intracellular redox state appears to be a necessary and sufficient modulator of the balance between self-renewal and differentiation in dividing oligodendrocyte-type-2 astrocyte progenitor cells. The intracellular redox state of freshly isolated progenitors allows prospective isolation of cells with different self-renewal characteristics. Redox state is itself modulated by cell-extrinsic signaling molecules that alter the balance between self-renewal and differentiation: growth factors that promote self-renewal cause progenitors to become more reduced, while signaling molecules that promote differentiation cause progenitors to become more oxidized. Moreover, pharmacological antagonists of the redox effects of these cell-extrinsic signaling molecules antagonize their effects on self-renewal and differentiation, indicating that cell-extrinsic signaling molecules that modulate this balance converge on redox modulation as a critical component of their effector mechanism.

Figure 1

Intracellular redox state of freshly isolated cells predicts self-renewal and differentiation in vitro. Analysis of clones was performed on freshly isolated immunopurified O-2A progenitors after cell sorting according to redox-sensitive Rosamine fluorescence. Rosamine^high cells represent a population with greater oxidative load than Rosamine^low cells. After 5 days in basal division conditions, Rosamine^low progenitors had a greater tendency to undergo self-renewing divisions than Rosamine^high cells, which in all but a few clones showed signs of rapid differentiation. Rosamine^high cells grown in medium supplemented with NAC, however, divided extensively and thus had not irreversibly committed to differentiation at the time of isolation.

Figure 2

Oxidizing agents promote differentiation of dividing O-2A progenitor cells, whereas NAC promotes self-renewal. Purified O-2A progenitor cells were grown at clonal density with PDGF ± redox-active drugs. After 5 days, 100 randomly chosen clones were analyzed for each condition. Histograms show the number of oligodendrocytes per clone along the x axis, the number of progenitors per clone (y axis), and the number of clones of each composition (z axis). Here, and in Fig. 4, a representative experiment is shown; each experiment was repeated at least three times with similar results. Progenitor cells treated with NAC exhibited more self-renewal as evidenced by larger clone sizes and a greater representation of progenitors per clone. In contrast, cells exposed to the pro-oxidants BSO or t-BuOOH exhibited extensive differentiation and less self-renewal.

Figure 3

Cell-signaling molecules that enhance self-renewal alter intracellular redox to a more reduced state, whereas those that promote differentiation alter intracellular redox to a more oxidized state. Cells were treated with various factors as indicated for 18 h, labeled with Rosamine, and analyzed by flow cytometry. Cells exposed to NT-3 or bFGF displayed less aggregate fluorescence, indicating a more reduced intracellular environment. Cells treated with TH or BMP-4 were on average more fluorescent, indicating increased oxidizing environments. Effects of NT-3 and TH on redox state were antagonized by treatment with BSO or NAC, respectively. Data are presented as the geometric means of triplicate analyses of multiple experiments, with data normalized to values obtained in the presence of PDGF alone.

Figure 4

NAC inhibits TH-mediated induction of differentiation, whereas BSO inhibits NT-3-mediated promotion of self-renewal. The ability of NT-3 to promote self-renewing divisions was blocked in the presence of BSO. After 5 days, the pattern of differentiation and self-renewal of clones treated with BSO + NT-3 resembled the profile exhibited in basal division conditions (compare with Fig. 2). Conversely, cells exposed to TH + NAC showed a profile of self-renewal similar to that seen in basal division conditions (see Fig. 2), in contrast with cultures supplemented with TH alone.