Embryonic stem cell-derived hematopoietic stem cells

Abstract

Despite two decades of studies documenting the in vitro blood-forming potential of murine embryonic stem cells (ESCs), achieving stable long-term blood engraftment of ESC-derived hematopoietic stem cells in irradiated mice has proven difficult. We have exploited the Cdx-Hox pathway, a genetic program important for blood development, to enhance the differentiation of ESCs along the hematopoietic lineage. Using an embryonic stem cell line engineered with tetracycline-inducible Cdx4, we demonstrate that ectopic Cdx4 expression promotes hematopoietic mesoderm specification, increases hematopoietic progenitor formation, and, together with HoxB4, enhances multilineage hematopoietic engraftment of lethally irradiated adult mice. Clonal analysis of retroviral integration sites confirms a common stem cell origin of lymphoid and myeloid populations in engrafted primary and secondary mice. These data document the cardinal stem cell features of self-renewal and multilineage differentiation of ESC-derived hematopoietic stem cells.

Fig. 1.

Characterization of ESC-derived hemangioblast and hematopoietic progenitors from an inducible Cdx4 cell line. (A) Quantification of blast colony-forming cells (BL-CFCs). A total of 3 × 10⁴ EB cells harvested on day 3.2 of differentiation from an inducible Cdx4 cell line were plated in blast-colony forming media in the absence or presence of doxycycline (dox), and colonies were counted 4 days after plating. A photograph of a representative blast colony is shown (Inset). (B) Methylcellulose colony-forming potential of day 6 EB-derived cells plated in methylcellulose containing cytokines (M3434). Colonies were counted from day 5 to 10 after plating. Ery^P/Ery^D, primitive/definitive erythroid; GEMM, granulocyte, erythroid, macrophage, megakaryocyte multilineage; GM, granulocyte macrophage; Mac, macrophage; Mast, mast cell. (C) Flow cytometric analysis of c-kit and CD41 on day 6 EBs. (D) Inducible Cdx4 ESC were treated with doxycycline from days 3 to 6 of EB formation and cultured on OP9 cells in the absence or presence of doxycycline. Fold increase of cell number on day 18 of OP9 culture was calculated relative to the initial cell number. (E) Relative expression levels of fetal (β-H1) and adult hemoglobin (β-major) before and after OP9 expansion by real-time RT-PCR analysis. (F) Relative expression levels of genes specific to different hematopoietic and lymphoid development pathways in Cdx4-induced or HoxB4-induced ESC-derived hematopoietic progenitors 15 days after OP9 expansion.

Fig. 2.

Hox gene expression profile in hematopoietic populations isolated from EBs by flow cytometry, determined by quantitative real-time RT-PCR analysis. (Upper) Flk1⁺ cells from day 4 EBs with (+dox) or without (-dox) Cdx4 induction from days 2 to 4 of EB differentiation. (Lower) CD41⁺ cells from day 6 EBs with (+dox) or without (-dox) Cdx4 induction from days 3 to 6 of EB differentiation.

Fig. 3.

Donor cell chimerism and CFU-S formation in mice engrafted with cdx4/hoxB4-modified cells. Mice were killed from days 6 to 12 and their spleens analyzed for CFU-S formation; the picture (Lower Right) shows a representative d12 spleen after fixation.

Fig. 4.

Donor cell chimerism and multilineage engraftment in irradiated primary and secondary mice. (A) Donor chimerism (%GFP⁺) in peripheral blood of mice engrafted with HoxB4 or Cdx4/HoxB4 modified hematopoietic populations differentiated from ESCs >22 weeks after transplantation. (B) Flow cytometry analysis of peripheral blood cells expressing either myeloid antigens (Gr-1, M) or lymphoid antigens (CD3/B220, L). Number of mice analyzed at each time point is indicated. (C) Donor chimerism in peripheral blood of secondary animals. Bone marrow (BM) from primary recipients engrafted at least 12 weeks was transplanted into secondary recipients. (D) Myeloid-lymphoid reconstitution of splenocytes from secondary animals. Error bars represent standard deviation. 1^ry, primary; 2^ry, secondary.

Fig. 5.

Clonal analysis of hematopoietic populations of mice engrafted with ESC-derived HSCs, as determined by Southern hybridization analysis of retroviral integration sites. (A) Structure of the retroviral vector MSCV-HoxB4-ires-GFP. Probes used in Southern hybridization analysis are indicated. (B Left) Southern analysis of fractionated myeloid and lymphoid populations from primary (1^ry) and unrelated secondary (2^ry) engrafted mice, showing multiple comigrating fragments. (B Right) Bone marrow and spleen cells from two primary engrafted animals and comparable tissue from the corresponding secondary animals, showing comigrating fragments. (C) Southern analysis of hematopoietic tissues from one primary and two corresponding secondary recipients engrafted with ESC-HSCs: spleen (S), BM (B), Gr1⁺ BM cells (B/G), Gr1⁺ splenocytes (S/G), and CD3⁺ or B220⁺ splenic lymphocytes (S/L). Mye/Lym represents the ratio of Gr-1⁺ cells to CD3⁺ and B220⁺ populations in corresponding sample, as determined by flow cytometry. Relative DNA level was calculated by comparing endogenous HoxB4 (endog) with control (DNA isolated from Ainv15 ES cells). Proviral copy number was calculated by comparing the level of proviral HoxB4 (Rv-HoxB4) with endogenous HoxB4 level. Samples reflect Cdx4/HoxB4-engrafted cells, except the third and fourth lanes in B Left, which represent HoxB4-treated cells. #, fragments detected only in primary recipients; ^*, fragments unique to secondary engrafted animals; ⁁, fragments detected predominantly in one lineage.