p21 and p27 mRNA expression during clonal culture of CD34⁺ cells. Human umbilical cord CD34⁺cells were incubated in methylcellulose culture containing cytokines or in suspension culture as described in Materials and Methods. From day 9 to 18, the indicated colonies were harvested for RNA extraction and cytosmeared for lineage confirmation (see Table 2). Purified CD34⁺ cells and cells in suspension culture of CD34⁺ cells were harvested for RNA extraction. RT-PCR was performed as described in Materials and Methods and each PCR product was separated on a 4.5% polyacrylamide gel followed by autoradiography. (A through E) Autoradiographs of RT-PCR products. Arrows indicate the PCR products corresponding to p21, p27, and β-actin. (F through J) p21 and p27 mRNA expression levels in the indicated colonies are standardized to β-actin determined by densitometry and are plotted over time in culture. The mean ± SE (bar) is shown. (○) p21; (•) p27. Numbers of colonies analyzed are denoted in parentheses. Seven cord blood samples were used for these studies.