Development of hESC-derived hematopoietic progenitor cells with expression of functional Notch-1. (A) Time course of hematopoietic progenitor cell production by hESCs. Coculture of undifferentiated hESCs with S17 stromal cells for the indicated number of days was followed by flow cytometric analysis of surface expression of CD34 and CD45. These results demonstrate surface antigen expression both on the unsorted cell population and the enriched CD34⁺ cell population. (B) hESC-derived cells were harvested after 14 days of culture on S17 stromal cells and analyzed by flow cytometry for expression of CD34 and Notch-1. The data were electronically gated to distinguish CD34⁺ cells (solid line) from CD34⁻ nonhematopoietic cells (dashed line). Isotype control staining is also indicated (filled histogram). (C) After 20 days of culture on S17 cells, total RNA was prepared from unsorted hESC/S17 cells or from the indicated populations of CD34⁺, CD34⁻, CD45⁺, or CD45⁻ cells. cDNA was prepared by reverse transcription, and expression of Notch-1, HES-1, and Actin was analyzed by PCR.

B-cell–promoting culture of UCB- and hESC-derived hematopoietic progenitors. (A) CD34⁺ UCB cells (○, ▵, □) proliferate in B-cell promoting culture but CD34⁺ hESC-derived cells (●, ▴, ■) do not. Hematopoietic progenitors were seeded into coculture on MS-5 stromal cells in RPMI 1640 containing 10% FBS, 1% P/S, and 10 ng/mL each of SCF and G-CSF (Reg: ○,●), or additionally supplemented with 10 ng/mL IL-7 (Reg + IL-7: ▵, ▴), or with 10 ng/mL IL-7 and Flt3L (Reg + IL-7 + Flt3L: □, ■). Hematopoietic cells were harvested after 13, 20, or 35 days and live cells counted to determine proliferation. (B) CD34⁺ UCB cells form B cells in MS-5 culture but CD34⁺ hESCs do not. Cells cultured for 35 days on MS-5 cells plus SCF and G-CSF were collected and analyzed for expression of human CD45 and CD19 by flow cytometry. Similar phenotypic results were seen with the other cytokine conditions in panel A.

The effect of cytokines on lymphoid lineage differentiation in vitro. (A) Addition of SCF, IL-3, and IL-15 promotes increased NK-cell differentiation. CD34⁺ hESC/S17 (left panels) and CD34⁺ UCB (right panels) cells were cocultured with OP9-DL1 stromal cells for 30 days. Cultures were supplemented with exogenous IL-7 and Flt3L (T-cell conditions) or with IL-7, Flt3L, SCF, IL-3, and IL-15 (NK-cell conditions). Cells were harvested and analyzed by flow cytometry for expression of CD4, CD8, and CD56. (B) To determine other sources of cytokines that could stimulate hESC-derived cells, the indicated mouse stromal cell lines were analyzed by RT-PCR for expression of the cytokines Scf, Flt3L, IL-7, and β-actin.

Development of T cells in vitro from UCB- but not from hESC-derived hematopoietic progenitor cells. (A) CD34⁺ UCB cells or (B) CD34⁺ hESC-derived cells were cocultured with OP9-DL1 or OP9-GFP stromal cells. Cells were harvested at the indicated time points and analyzed by flow cytometry for surface expression of CD4, CD8, CD7, and CD1a. (C) FTOC culture of UCB- and hESC-derived hematopoietic progenitors. CD34⁺ cells from UCB and hESC/S17 sources were cultured for 12 days in mouse fetal thymic lobes. Cultured cells were analyzed by flow cytometry for expression of CD4 and CD8 using antibodies specific for human proteins. (D) CD34⁺ CD45⁺ hESC-derived progenitors were cocultured with OP9-DL1 for 14 days and analyzed by flow cytometry for expression of CD4, CD8, CD11c, CD123, CD33, CD45, and CD56.

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.