Hematopoiesis and immunity of HOXB4-transduced embryonic stem cell-derived hematopoietic progenitor cells

Abstract

The ability of embryonic stem (ES) cells to form cells and tissues from all 3 germ layers can be exploited to generate cells that can be used to treat diseases. In particular, successful generation of hematopoietic cells from ES cells could provide safer and less immunogenic cells than bone marrow cells, which require severe host preconditioning when transplanted across major histocompatibility complex barriers. Here, we exploited the self-renewal properties of ectopically expressed HOXB4, a homeobox transcription factor, to generate hematopoietic progenitor cells (HPCs) that successfully induce high-level mixed chimerism and long-term engraftment in recipient mice. The HPCs partially restored splenic architecture in Rag2(-/-)gamma(c)(-/-)-immunodeficient mice. In addition, HPC-derived newly generated T cells were able to mount a peptide-specific response to lymphocytic choriomeningitis virus and specifically secreted interleukin-2 and interferon-gamma upon CD3 stimulation. In addition, HPC-derived antigen presenting cells in chimeric mice efficiently presented viral antigen to wild-type T cells. These results demonstrate for the first time that leukocytes derived from ES cells ectopically expressing HOXB4 are immunologically functional, opening up new opportunities for the use of ES cell-derived HPCs in the treatment of hematologic and immunologic diseases.

Figure 1

Differentiation of HOXB4-transduced ES cells. (A) Representative flow cytometric analysis and phase-contrast microscopy of HOXB4-transduced ES cells under hematopoietic differentiation medium show increased expression of GFP and CD45 as their differentiation progressed from day 0 to day 26. The fully differentiated HPCs showed various sizes of single cells at day 26. Numbers indicate percentages of cells within the culture. Original magnification ×200 for photographs. Numbers on plots are percentages of total cells in that quadrant. (B) Total number of viable cells was counted at different days after differentiation. HOXB4-transduced ES cells survived and eventually robustly expanded up to nearly 100-fold after 26 days of differentiation. In contrast, nontransduced control CCE-GFP ES cells failed to thrive and progressively regressed in viable cell numbers. Days 1 through 6 represent period of EBs formation.

Figure 2

HPCs derived from HOXB4-transduced ES cells contribute to multilineage hematopoiesis in Rag2^−/−γ_c^−/− mice. (A) Flow cytometric analysis of peripheral blood, spleen, and bone marrow of a representative Rag2^−/−γ_c^−/− mouse 56 days after HPC transplantation (n = 5). Numbers represent percentages of cells to the total GFP-expressing cell population. The majority of the HPC-derived cells were Gr-1 positive and therefore myeloid. (B) Flow cytometric analysis of hematopoietic cells in peripheral blood, spleen, and bone marrow cells of chimeric Rag2^−/−γ_c^−/− mice transplanted with either HPCs (56 days after transplantation [Tx]) or with marrow cells of GFP transgenic mice (60 days after transplantation). Donor-derived cells were predominantly Gr-1 positive after HPC transplantation compared with B220-positive–expressing cells after BMT (BM Tx). Error bars represent SD.

Figure 3

HPCs populate lymphoid tissues. Histologic cryosections of the spleen, liver, and thymus were stained for GFP using an anti-GFP monoclonal antibody and examined under the Multi photon/confocal microscope. (A) GFP-expressing cells could be detected in both spleen and liver early after HPC transplantation, diminishing in the liver with time. Scale bars represent 50 μm. (B) GFP-positive cells remained detectable in the thymus 56 days after transplantation. At each time point, n ≥ 3 mice. Scale bar represents 50 μm.

Figure 4

HPC engraftment restores splenic follicular structure in Rag2^−/−γ_c^−/− mice. (A) Histologic splenic sections of WT B6, chimeric, and nonchimeric Rag2^−/−γ_c^−/− mice were examined. The spleen from a nonchimeric Rag2^−/−γ_c^−/− mouse shows complete loss of follicular structure and hypocellularity in the periarteriolar region. The chimeric Rag2^−/−γ_c^−/− mouse (56 days after transplantation) shows follicular reconstitution as well as hypercellularity around the periarteriolar regions, comparable to the spleen of the mouse reconstituted with WT B6 bone marrow cells. Control mice were nontransplanted B6 mice. The bottom panels show the histologic sections at higher magnification. (B) Similarly, GFP staining in the HPC-induced chimeric Rag2^−/−γ_c^−/− mouse showed that GFP-positive donor-derived cells clustered in follicular form within the spleen. The bottom panel shows the same section at higher magnification. Scale bars represent 50 μm. (C) To further demonstrate the phenotype of the transplant-derived cells, splenic tissues of recipient Rag2^−/−γ_c^−/− mice were stained for Gr-1 and B220. Mice that received the defective Rag2^−/−γ_c^−/− bone marrow showed poor recovery of the follicles and a higher percentage of Gr-1–expressing cells but no B220-positive cells. However, both mice receiving either HPCs or WT bone marrow showed restoration of the follicular structure. The HPC-derived cells were predominantly Gr-1–positive cells, in contrast to the high levels of B220-positive cells in the mouse engrafted with B6 bone marrow cells. Both Gr-1–positive and B220-positive cells are stained red. Scale bars represent 50 μm.

Figure 5

Chimeric Rag2^−/−γ_c^−/− mice generate peptide-specific T cells after LCMV infection, and HPC-derived APCs are capable of presenting viral-specific peptide to T cells. (A) Splenocytes of viral-infected mice were short-term incubated in the presence or absence of LCMV peptide NP396-404. CD8-positive T-cell responses were determined by intracellular IFN-γ staining, and IFN-γ–secreting cells were detected in WT and chimeric mice but not in control nonchimeric Rag2^−/−γ_c^−/− mice. IFN-γ–positive CD8-positive T cells were nearly undetectable in all controls. Representative flow cytometric plots are shown for each group of mice (n = 3). (B) The IFN-γ–positive cells in the chimeric Rag2^−/−γ_c^−/− mice were also GFP positive, suggesting they were derived from HPCs. The numbers represent percentages of cells in each quadrant. Numbers in plots are percentages of total cells in that quadrant. (C) HPC-derived APCs pulsed with the LCMV NP396-404 peptide, control OVA257-264 peptide, and nonpulsed cells were coincubated with WT T cells isolated from LCMV-infected WT mice for 6 hours, respectively. T-cell responses were determined by intracellular staining of IFN-γ and analyzed by FACS. The HPC-derived APCs significantly presented viral specific peptide but not control peptide to primed T cells (n = 3). Error bars represent SD.

Figure 6

HPC-derived T cells respond to CD3 signaling. T cells were isolated from the splenocytes of chimeric Rag2^−/−γ_c^−/− mice (n = 3) and WT 129/SvJ control mice (n = 2). The T cells were subsequently cultured in the absence (▭) or presence (▬) of plate-bound antibodies against CD3 and CD28 for 24 to 36 hours. The supernatant of cultures were then collected and assessed for IL-2 (A) and IFN-γ (B) production by ELISA. Error bars represent SD.

Figure 7

Serum levels of TNP-specific antibodies after immunization and augmentation of T- and B-cell expansion. (A-C) WT, chimeric, and nonchimeric Rag2^−/−γ_c^−/− mice were immunized with either TNP-KLH or TNP-LPS. Serum levels of antibodies were determined by ELISA in duplicates and the results presented as arbitrary units. Serum levels of TNP-specific antibodies in chimeric and nonchimeric mice immunized with TNP-KLH are represented in A, and those to TNP-LPS are in B. Serum levels of TNP-specific antibodies in WT B6 mice immunized with either TNP-KLH or TNP-LPS are shown in C. Error bars represent SD. (D) Representative flow cytometric data from peripheral blood of a chimeric mouse without vaccination showing low T- and B-cell levels (left panel). In contrast, after vaccination, both T- and B-cells show a huge expansion (right panel). In addition, 2 populations of GFP^dim (36% of GFP-positive) and GFP^high (54% of GFP-positive), marked with circles were identified within the GFP-positive cell population of B220-positive cells. Numbers represent percentages of the proportion of the lymphocytes to the total GFP-expressing cells.