Cell size and RNA content correlate with cell differentiation and proliferative capacity of rat keratinocytes

Keratinocytes from rat skin were separated according to their size in a specially designed unit-gravity sedimentation chamber. The fractions obtained with this technique showed clear morphological differences, and analysis of size distribution confirmed that size was the criterion for separation. Simultaneous DNA and RNA staining of the fractions with acridine orange and subsequent flow cytometric analysis enabled one to classify cells into resting, proliferating, and differentiating stages. Cell size was not directly correlated with proliferation in situ as determined with acridine orange flow cytometry, nor with proliferative capacity in culture as assayed by BrdU/Hoechst flow cytometry. The smallest cells, exhibiting low DNA and RNA content, which do not proliferate in vivo, required a prolonged period of serum stimulation in vitro to initiate RNA and DNA synthesis. Cells of intermediate size exhibited early RNA synthesis and maximal proliferative capacity, whereas the largest cell population displayed no RNA synthesis in culture and the least proliferative capacity. In conclusion, these results suggest that RNA synthesis early after serum stimulation, in addition to a specific, optimal cell size, correlates with the proliferative capacity of keratinocytes in cell culture.