hESC-derived hematopoietic cells constitutively express transcription factors that promote NK-cell development from lymphoid progenitor cells. (A) UCB CD34⁺ cells, undifferentiated hESCs, and indicated differentiated hESC-derived cell populations were analyzed for expression of E2A, ID1, ID2, ID3, ID4, and ACTIN by RT-PCR. hESC/S17 represents hESC allowed to differentiate on S17 stromal cells for 14 days and analyzed either as an unsorted population, or sorted for CD34⁺CD45⁺, CD34⁺ Flk1⁻, or CD34⁺ Flk1⁺ cells as indicated. (B) Gene expression in hESC- and UCB-derived hematopoietic progenitor cells during NK differentiation. CD34⁺ hESC/S17 cells at 16 days of differentiation (left panels) and CD34⁺ UCB (center panels) cells were cultured in NK supporting conditions for indicated numbers of days, then analyzed at the indicated days for expression of ID1, ID2, ID3, RAG-1, CD122, SLUG, and ACTIN genes by RT-PCR. (C) Q-RT-PCR of ID2 and ID3 expression from hESCs cocultured with S17 stromal cells in standard differentiation conditions (□, RPMI + 15% FBS) or with serum-free medium containing BMP-4 (■, StemPro + BMP-4). Cells were harvested after the indicated number of days and analyzed by Q-RT-PCR for ID2, ID3, and ACTIN expression. cDNA content was normalized according to actin levels, and expression of ID2 and ID3 was calculated relative to day-0 expression levels. Data from 1 of 2 replicate studies are shown. (D) Q-RT-PCR analysis of E2A-responsive genes LAT, IL7Rα, and IL1a in hESC/S17 cells at 16 days of differentiation (), sorted hESC-derived CD34⁺CD45⁺ cells (), and CD34⁺ cells from UCB (■). Results are presented as relative gene expression normalized to UCB CD34⁺ cells.