Differentiation potential of bipotent CD235a⁺/CD41a⁺ PEM. (A) Representative flow cytometric histogram demonstrating the CD235a⁺/CD41a⁺ immunofluorescent staining pattern of cord blood CD34⁺ cells transduced with the Y* CGS lentiviral vector and expanded for 14 days in 100 nM AP20187. Gates were set based on staining controls. The percent of cells in each quadrant are indicated. (B) Relative progenitor content of CD235a/CD41a subpopulations. Cells were sorted from day 14 to 21 cord blood CD34⁺ cultures expanded with the Y* CGS lentiviral vector based on presence or absence of CD235a and CD41a expression, and plated in methylcellulose cultures supplemented with a broad cytokine cocktail. Data represent the mean ± SD from 3 independent experiments. P values are based on the t test vs the unsorted control. (C) Example of a mixed progenitor colony generated from sorted CD235a⁺/CD41a⁺ PEM, showing the presence of both erythroid bursts (dark globules at bottom) and nonerythroid cells (white colony at top). (D) Differentiation potential of PEM. CD235a⁺/CD41a⁺ PEM were sorted from day 14 cord blood CD34⁺ Y* CGS expansion cultures and transferred to media supporting either erythroid (Epo + SCF) or megakaryocytic (TPO, IL-6, IL-9, SCF) differentiation. After 6 days, the cells were collected, prepared as cytospins, and stained by Wright-Giemsa. Left, sorted cells before secondary culture characterized morphologically as erythroid precursors; middle, representative of erythroid cultures characterized morphologically as enucleated red blood cells and a few pre-erythrocytes; and right, representative of megakaryocyte cultures characterized morphologically as megakaryocytes exhibiting mono- and polylobular nuclei and surrounded by a halo of platelets, and confirmed as CD41a⁺/CD42b⁺ by immunofluorescent staining and flow cytometry.