In vitro differentiation of hESC toward hematopoietic lineage. (a) Changes in hematopoietic marker expression after in vitro culture of EGFP⁺ H1 cells on OP9 cells. Cells were analyzed by flow cytometry and gated on EGFP⁺ cells (which typically ranged between 70% and 90% of total live cells). Expression of each marker was determined at the indicated times. (b) Flow cytometric analysis of hematopoietic marker expression in the CD34⁺ cell population (typically <5% of total live cells). (c) hESC-derived hematopoietic progenitor cells give rise to erythroid (Upper) and myeloid (Lower) colonies that express EGFP, as viewed by phase-contrast (Left) and fluorescent (Right) microscopy after 10 days of differentiation on OP9 cells and 14 days of growth in methylcellulose containing erythropoietin, stem cell factor, granulocyte–macrophage colony-stimulating factor, and IL-3.

Phenotypic analysis of hESC-derived thymocytes. (a) To distinguish endogenous HLA-A2⁻ and hESC-derived HLA-A2⁺ cells, thymocytes from control mice (Left) and SCID-hu mice into which hESC-derived CD34⁺ cells had been transferred (Right) were stained and gated based on the expression of CD45 and EGFP. MFI, mean fluorescence intensity. Cells within each gate were subsequently analyzed for the expression of HLA-A2. (b) CD45⁺, EGFP⁺ cells from the Thy/Liv implants from the RAG-hu mice transferred with hESC-derived CD34⁺ cells (Upper) and CD45⁺ cells from the implants of the control animals (Lower) were gated and analyzed for the presence of the indicated T cell surface markers. Region markers are based on isotype controls.

Costimulation of EGFP⁺ thymocytes after 5 weeks of differentiation in vivo. Cells were cultured in medium alone (unstimulated) or in the presence of anti-CD3 and anti-CD28 monoclonal antibodies (costimulated), and EGFP⁺ cells were analyzed for expression of CD25 by flow cytometry.

Genetic modification of undifferentiated hESC. The hESC line H1 was transduced with the EGFP expression lentiviral vector pSIN18.cPPT.hEF1α.EGFP.WPRE and selected for homogenously green colonies. Upper and Lower illustrate the same colonies in each column. (a and b) Phase-contrast (a) and fluorescent (b) images of an EGFP-labeled H1 colony at passage 71. (c, e, and f) The undifferentiated phenotype and transgene expression were maintained as shown by the expression of the early stem cell markers Oct-4 (c) and alkaline phosphatase (f), as well as EGFP (e), at passage 122. (d) DAPI stain.

In vivo T lymphoid differentiation of H1-derived progenitor cells expressing EGFP. (a) Schematic representation of differentiation protocol. (b and c) Flow cytometry profiles of cells derived from irradiated SCID-hu Thy/Liv mice 3 weeks (b) (denoted week 3) and 5 weeks (c) (denoted week 5) after transplantation with no cells (Mock) (Upper Left), sorted CD34⁺ progenitor cells (Upper Center), or sorted CD133⁺ progenitor cells (Upper Right) that were derived from HI cells previously cultured on OP9 cells for 11 days (denoted by d11). SCID-hu mice were biopsied on two successive occasions, and thymocytes were analyzed for expression of EGFP, CD45, CD3, CD4, and CD8 by multicolor flow cytometry. Cells were analyzed by gating on live cells (forward vs. side scatter), then gating on CD45⁺ to identify human hematopoietic cells (not shown). (Upper) Live, CD45⁺ cells were then analyzed for CD3 and EGFP. Numbers in each corner represent the percentage of cells in each quadrant, and the gate drawn through the upper and lower right quadrants denotes EGFP⁺ cells, with the percentage of cells given within the gate. (Lower) CD4 vs. CD8 profiles of the EGFP⁺ population, determined by gating.

hESC-derived thymocytes maintain expression of the reporter transgene in vivo. CD45^dim HLA-A2⁺ thymocytes from SCID-hu mice into which hESC-derived CD34⁺ cells had been transferred (Center) were analyzed for the expression of EGFP (Right). (Left) Lack of gated population in control mice receiving no hESC-derived progenitors.