Most of the factors (peptide hormones, steroids, vitamins, etc.) that have been shown to affect the erythroid differentiation process are present at trace level in numerous biological fluids (mainly sera) used in various culture systems. Furthermore, some of these factors are produced in an autocrine fashion. Therefore, the role of these factors usually has to be analyzed only in defined systems specifically deprived in a given factor (using specific inhibitors, depleted sera, neutralizing antibodies, as well as dominant-negative isoforms).
Furthermore, most of the effects we observed relied upon a cooperative action of at least two factors. Studying one type of factor without controlling the effect of the other might result in pretty diverse biological readouts.
In the case of thyroid hormone action, the final potential source for confusion stems from redundancy. Indeed, we have shown that the potential sources of hormones activating both T3Ra and RARa have to be neutralized in order to obtain a significant decrease in the differentiation ability of the progenitors (41). Such a co-depletion was accomplished by preincubating the complete differentiation medium with the anti-T3 (monoclonal antibody from BioGenesis) at 0.3% (v/v) and the RARa-specific antagonist Ro41-5253 at 10-7 M at 4°C for 1 h under constant agitation. Under those conditions, a 40% decrease in the percent of benzidine-positive cells should be observed (41).
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